RU2268584C2 - Barley of reduced starch ii synthase (ssii) activity - Google Patents

Abstract

FIELD: biotechnology, food processing industry.

SUBSTANCE: barley plants are mutated in SSII gene to reduce SSII activity. Such barley corn has starch structure with. Moreover said corn optionally has relatively high β-glucan content. Starch is characterized with decreased gelatinization viscosity, low crystallinity, and high levels of lipid-bonded starch of V-form crystallinity.

EFFECT: barley plants having reduced amylopectin content and relatively high amylose content.

33 cl, 39 dwg, 10 tbl, 2 ex

Description

This invention relates to a barley plant with reduced starch synthase II (SSII) enzyme activity leading to starch having a reduced amylopectin content. In addition, this invention relates to starch and grain, as well as to food products derived from them.

BACKGROUND OF THE INVENTION

One of the discoveries in the science of nutrition is the fact that resistant starch is important for intestinal health, in particular for colon health. The beneficial effects of resistant starch are the result of providing nutrition to the large intestine, where the intestinal microflora receives an energy source that undergoes fermentation to form, among other things, short chain fatty acids. These short chain fatty acids provide nutrients for colonocytes, enhance the uptake of certain nutrients through the colon, and promote the physiological activity of the colon. Generally, if resistant starches or other dietary fiber is not provided, the colon is relatively inactive in metabolism.

In recent years, there has been a direction in the search for the development of resistant starches from various sources aimed at intestinal health. Accordingly, high amylose starches are found in some cereals, such as corn, for use in food products as a means of promoting intestinal health.

The physical structure of starch can have an important effect on the nutritional and technological properties of starch for food. Some characteristics can be considered as an indicator of the structure of starch, including the distribution of amylopectin chain lengths, the degree of crystallinity and the presence of crystallinity forms, such as the V-complex form of crystallinity of starch. Forms with such characteristics can also be considered as an indicator of the nutritional or technological properties of food products containing these starches. Thus, the short chain length of amylopectin can be an indicator of low crystallinity and weak gelatinization, and in addition, it is believed that it correlates with reduced retrogradation of amylopectin. In addition, it is believed that the distribution of the lengths of shorter chains of amylopectin reflects the organoleptic properties of food products in which this starch is included in significant quantities. The reduced crystallinity of starch may also be an indicator of the reduced gelatinization temperature of starch, and moreover, it is believed that it is associated with improved organoleptic properties. The presence of V-complex crystallinity or a lipid otherwise associated with starch will increase the level of resistant starch and, therefore, dietary fiber.

Barley lines with high amylose starches have been identified previously. The result was only relatively small increases in the amylose content to a maximum value of about 45% of the total starch, such as in the barley variety known as High Amylose Glacier (AC38). Although starches with a high amylose content of this type are useful, starch with a higher amylose content is still preferred, and some other types of cereals are grown to produce starches with a higher amylose content at 90 percent range. They are very resistant to enzymatic hydrolysis and bring great health benefits.

There is a problem in the development of high amylose starches, since the known high amylose starches also have a high gelation temperature. The gelatinization temperature reflects the grinding energy required to process such foods. Therefore, for processing grain or flour for the production of food products from such grains or starches, higher temperatures are usually required. Therefore, as a rule, products having high amylose starches are more expensive. Similarly, from the consumer's point of view, longer periods of time and higher temperatures may be required to prepare these manufactured foods or to prepare food from flour having high amylose starches. Therefore, there is considerable inconvenience in providing high amylose starches in foods.

Another nutritional component of cereals and, in particular, barley are β-glucans. β-Glucans consist of glucose units linked by β (1-4) and / or β (1-3) glycosidic bonds and are also not destroyed by human digestive enzymes, which makes them suitable as a source of dietary fiber. β-Glucans can be partially subjected to enzymatic hydrolysis by endogenous colonic bacteria, during the fermentation of which short-chain fatty acids (mainly acetate, propionate and butyrate) are formed, which are useful for mucous membrane cells lining the intestines and colon (Sakata and Engelhard Comp. Biochem. Physiol. 74a: 459-462 (1983)).

In addition, the absorption of β-glucan has the effect of increasing the secretion of bile acids, leading to a decrease in total serum cholesterol and low density lipoproteins (LDL) with a decrease in the risk of coronary heart disease. Similarly, β-glucans act by attenuating blood glucose shifts that occur after eating. Both of these effects are believed to be based on increasing the viscosity of the contents of the stomach and intestines.

The composition of food products containing starches and the close interactions of these starches with other nutritional or other components can have a significant impact on the nutritional value of these food products or on the functional characteristics of these components in the preparation or structure of these food products.

Although modified starches or β-glucans, for example, can be used in foods that provide functionality not normally provided by unmodified sources, such processing tends to either modify other important components or is undesirable due to the processes involved in the modification. Therefore, it is preferable to offer sources of components that can be used in unmodified form in food products.

Barley variety MK6827 is available from the Barley Germplasma Collection (USDA-ARS National Small Grain Germplasma Research Facility Aberdeen, Idaho 831290 USA). The MK6827 grain is wrinkled and has a strongly colored husk and an oblong shape, and in the hands of the inventors this grain is very difficult to process, including the fact that it is very resistant to grinding. The properties of MK6827 grain have not been previously characterized, the nature of the mutation has not been established, and it is not considered suitable for food production.

SUMMARY OF THE INVENTION

This invention is the result of isolation and characterization of the SSII mutant of barley plants, the grain of which is found to contain starch, which has a low amylopectin content and, therefore, high relative levels of amylose, and therefore has elevated levels of dietary fiber.

The grain of this mutant and grain from crossing into some genetic backgrounds, in addition, has an increased level of β-glucan. The combination of increased levels of β-glucan and resistant starch, contributing to a high level of dietary fiber, the inventors consider unique to the present invention.

In addition, at least in some genetic backgrounds, it has been found that the grain from such mutants contains starch, which has high relative amylose levels and also has low gelatinization temperatures. The low swelling characteristics of such starch during and after gelatinization also have advantages in some applications of dietary and food technology.

In addition, it was found that the grain from such mutants contains starch, which has high relative amylose levels, and the detected amylose levels are higher than 50% of the starch content, which is a level that was never previously found in unmodified starch obtained from barley.

Starch of mutants and backcross lines originating from these mutants (to the extent to which these backcross lines have been tested) behaves as resistant starch with a modified structure, which is indicated by specific physical characteristics, including one or more than one of the group including the presence of a high relative content of amylose, physical inaccessibility due to the presence of a high content of β-glucan, altered morphology of the granules and the presence of starch-bound lipid And this modified structure also specifies a characteristic selected from one or more than one of the group comprising low crystallinity, reduced amylopectin chain length distribution and presence of appreciable starch associated lipid.

In addition, the grain obtained from these mutant barley plants can be easily used in food technology procedures.

The present invention, in one aspect, relates to starch obtained from a grain of a barley plant having a reduced level of SSII activity, said starch granules having a high amylose content due to a reduced amylopectin content.

The present invention, in another broader aspect, relates to a grain useful for the manufacture of food products obtained from a barley plant having a reduced level of SSII activity, wherein the starch of said grain has a high amylose content due to the reduced amylopectin content.

The present invention, in yet a broader aspect, relates to a barley plant with a reduced level of SSII activity, said barley plant being capable of producing grain, the starch of said grain having a high amylose content due to the reduced amylopectin content, said grain being suitable for food production.

Alternatively, the present invention relates to an isolated nucleic acid molecule encoding an SSII barley protein, said nucleic acid being capable of hybridization under stringent conditions with SEQ ID NO 1, or a cell carrying a replicable recombinant vector carrying said nucleic acid molecule. In yet another embodiment, the present invention relates to an isolated nucleic acid molecule capable of specific hybridization with SEQ ID NO 1.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

For a better understanding, the invention will be described below with reference to a number of examples.

Figure 1. Analysis of the molecular size distribution of starch, as determined by HPLC starch separation in 90% DMSO. (a) Himalaya, (b) AC38, (c) 342, (d) 292.

Figure 2. Photos showing the grain morphology of the mutant and parental lines, (a) Himalaya, (b) AC38, (c) 292, (d) Waxiro, (e) 342, (e) Tantangara, (g) MK6827, (h) Sloop. The grain sizes in length (L), width (W) and thickness (T) are illustrated in panel (a).

, 292

, Tantangara (s), AC38

, МК6827

и Himalaya (+).Figure 3. Analysis of the chain length distribution of various mutant and wild-type starches using FACE. (a) Normalized distribution of chain lengths, (b) comparison of distribution of chain lengths using a difference diagram. Samples were 342

, 292

, Tantangara (s), AC38

MK6827

and Himalaya (+).

, Namoi (Δ), AC38 (О), 342 (▽), 292

и МК6827

. Температурный профиль, используемый на протяжении профиля, указан непрерывной линией.Figure 4. RVA analysis of barley starch samples. Samples were Himalaya

, Namoi (Δ), AC38 (O), 342 (▽), 292

and MK6827

. The temperature profile used throughout the profile is indicated by a continuous line.

Figure 5. X-ray diffraction data for the mutant line and the wild-type line.

6. Microphotographs in a scanning electron microscope of the isolated barley starches, (a) Himalaya, (b) Waxiro, (c) AC38, (d) 292, (e) 342, (e) MK6827.

7. Loci on barley 7H chromosome showing the proximity of nud1 and sex6 loci. Chart according to GrainGenes (http://wheat.pw.usda.gov/) Barley morphological genes, 7H map, author; Franckowiak JD).

Fig. 8. Relationship between seed size and starch chain length distribution for 292 × Tantangara double haploid lines. The lines indicated by (+) gave the Himalaya PCR picture, and the lines indicated by (o) gave the result of PCR 292. Panel (A), the ratio of seed length to thickness plotted against the percentage of starch chains with DP from 6 to 11; panel (B), the mass of the seed plotted against the percentage of starch chains with DP from 6 to 11.

Fig.9. The barley SSII cDNA sequence (SEQ ID NO 1) from the Himalaya variety.

Figure 10. The structure of SSII genes from (1) T.tauschii (diploid wheat), (2) Morex barley variety. Lines in bold represent exons, and thin lines represent introns. A straight line below each example indicates a portion of gene sequences. The dashed line shows the plot of the SSII gene of barley from intron 7, which was not sequenced, but was determined by PCR analysis as approximately 3 kb. in length.

11. Comparisons of predicted SSII cDNAs from MK6827 (SEQ ID NO 2), Morex (SEQ ID NO 3) and 292 (SEQ ID NO 4) and Himalaya cDNA sequences (SEQ ID NO 1). The predicted sequences were obtained by identifying portions of genomic sequences present in the Himalaya SSII cDNA. The ATG start codon and wild-type stop codon are indicated, as are the additional stop codons present in MK6827 (#) and 292 (&), respectively.

Fig. 12. Comparison of amino acid sequences derived from genes encoding SSII from barley lines 292 (SEQ ID NO 7), Morex (SEQ ID NO 5), MK6827 (SEQ ID NO 8), Himalaya (SEQ ID NO 6). Additional stop codons at 292 and MK6827 are indicated by (&) and (#), respectively.

Fig.13. The position of the mutations in MK6827 (SEQ ID NO 2) and 292 (SEQ ID NO 4) in the barley SSII gene.

Fig.14. Development and use of PCR analysis for mutation 292. (a) Schematic representation of the SSII region from Himalaya amplified using ZLSS2P4 and ZLBSSIIP5 primers, (b) image of the region amplified from the SSII gene from 292 using ZLSS2P4 and ZLBSSIIP5, showing the absence of one site NIaIV, (c) agarose gel electrophoresis of NIaIV enzymatic hydrolysis products from barley; Track M; DNA marker ladder, lane 1: MK6827, lane 2: Himalaya, lane 3: Tantangara, lane 4: 292, lane 5: 342.

Fig.15. SDS-PAGE electrophoresis (electrophoresis in polyacrylamide gel with sodium dodecyl sulfate) starch granule proteins. Panel (A) 8% acrylamide (37.5: 1 acrylic / bis) SDS-PAGE gel subjected to electro-blotting and probing with SSII antibody obtained against purified granule-bound SSII protein from wheat. (B) 12.5% acrylamide (30: 0.135 acrylic / bis), silver stained. Migration of molecular weight standards of a certain mass (units are kD) are indicated on each side of the figure.

Fig.16. Schematic representation of DNA constructs designed for negative regulation of SSII expression after stable transformation of barley. (1) The SSII gene from nucleotide 1 to 2972 (see sequence in FIG. 9) is inserted between the promoter and the terminator in a sense orientation. (2) The SSII gene is inserted between the promoter and terminator in an antisense orientation from nucleotide 2972 to 1 (see sequence in FIG. 9). (3) A double construct in which the intron 3 of the barley SSII gene (between nucleotides 1559 and 2851) of the SSII Morex genomic sequence is inserted between exons 2 and 3 of the barley SSII cDNA from Himalaya (nucleotides 363-1157 in FIG. 9).

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Glycemic index. This is a comparison of the effects of test foods, such as white bread or glucose, on shifts in blood glucose concentrations. The Glycemic Index is a measure of the likely effect of food associated with the concentration of glucose in serum after a meal and the need for insulin for blood glucose homeostasis.

Resistant Starch. The sum of starch and starch enzymatic hydrolysis products that are not absorbed in the small intestine of healthy people, but enter the large intestine. Thus, resistant starch eliminates products that are digested and absorbed in the small intestine.

Resistant starches can be classified into four groups.

RS1 is physically inaccessible starch. Examples of this form of starch are formed where the starch is enclosed within a protein or similar matrix, either within a plant cell wall, or it may form due to partial grinding of grain or legumes after cooling.

RS2 resistant granules. These are usually raw starches, such as those made from raw potatoes or green bananas, some legumes and high amylose starches.

RS3 retrograded starches. These starches are formed by the thermal / wet processing of starch or starchy foods, such as those found in cooked and chilled potatoes, bread and corn flakes.

RS4 chemically modified. These starches are formed due to chemical modifications, such as substitution or crosslinking. This form of starch is often used in processed foods.

Dietary fiber. In this description, this is the sum of carbohydrates or enzymatic hydrolysis products of carbohydrates that are not absorbed in the small intestine of healthy people, but enter the large intestine. They include resistant starch, β-glucan and other soluble and insoluble carbohydrate polymers. The portion of carbohydrates that is at least partially susceptible to fermentation by resident microflora in the large intestine should be included.

Gelatinization is the destruction (interruption) of molecular order within a starch granule with concomitant and irreversible changes in properties such as granular swelling, crystallite melting, loss of birefringence, viscosity development and solubilization of starch.

This invention arose from the isolation and characterization of SSII mutant barley plants, the grain of which was found to contain starch, which has a reduced amylopectin content and, therefore, high relative amylose levels, and therefore has elevated levels of dietary fiber.

It has been found that such mutants possess a number of highly desirable characteristics, and it has been shown that crosses into various other genetic backgrounds retain at least some of these characteristics.

Mutant grain and grain from crosses in some genetic backgrounds, in addition, have an increased level of β-glucan. The combination of increased levels of β-glucan and high levels of dietary fiber, the inventors consider unique to the present invention.

In addition, it was found that, at least in some genetic backgrounds, the grain from such mutants contains starch, which has high relative levels of amylose and also has low gelatinization temperatures. The swelling characteristics of the gelatinization of such starch also have the advantage of being a low swelling, which is useful in some applications in diet and food technology.

In addition, it was found that the grain from such mutants contains starch, which has high relative amylose levels, and the detected amylose levels are higher than 50% of the starch content, which is a level that was never previously found in unmodified starch obtained from barley.

Starch of mutants and backcross lines to the extent to which they are tested behaves like stable starch with a modified structure, which is indicated by specific physical characteristics, including one or more than one of the group including the presence of a high relative amylose content, physical inaccessibility due to the presence of a high content of β-glucan, an altered morphology of the granules and the presence of a starch-bound lipid, and this characteristic is also indicated by a characteristic selected mated from one or more than one of the group comprising low crystallinity, reduced amylopectin chain length distribution and presence of appreciable starch associated lipid.

In addition, for this reason, the grain obtained from these mutant barley plants can be easily used in food technology procedures.

Grain from such mutants in one form preferably contains starch, which has high relative levels of dietary fiber, more specifically amylose, as well as elevated levels of β-glucan. The inventors consider the combination of increased levels of β-glucan and high levels of amylose unique to the present invention, and it provides a unique source of combination of β-glucan and resistant starch, which does not require, at least in the broader aspects of the invention, mixing β-glucan and soluble dietary fiber together or modifications to these components.

As far as the authors of the invention are aware, the barley plant of the present invention is the first open barley grain crop having elevated relative levels of dietary fiber in the form of resistant starch having elevated levels of amylose, which also has elevated levels of β-glucan, which are at a higher point of typical β levels -glucan or that exceed this level. Grains that have an even higher β-glucan content are a waxy phenotype and therefore have low levels of amylose.

It is known that there is a wide variety of β-glucan levels in barley ranging from about 4% to about 18% w / w barley, but more typically from 4% to about 8% (Izydorcyk et at. (2000) Journal of Agricultural and Food Chemistry 48, 982-989; Zheng et al. (2000) Cereal Chemistry 77, 140-144; Elfverson et al. (1999) Cereal Chemistry 76, 434-438; Andersson et al. (1999) Journal of the Science of Foods and Agriculture 79, 979-986; Oscarsson et al. (1996) J. Cereal Science 24, 161-170; Fastnaught et al. (1996) Crop Science 36, 941-946). Improved barley lines have been developed, for example Prowashonupana, which have from about 15% to about 18% w / w β-glucan but have the waxy phenotype. It is commercially available under the name Sustagrain ™ (ConAgra ™ Specially Grain Products Company, Omaha, Neb. USA).

The levels of β-glucan contemplated by this invention may depend on the genetic background at which the enzymatic activity of amylopectin synthesis is reduced. However, it is suggested that a decrease in the activity of amylopectin synthesis will have the effect of increasing the relative level of dietary fiber, which partially takes the form of amylose, and at the same time increasing the level of β-glucan. One explanation for the concomitant increase in β-glucan at elevated relative amylose levels is that such an increase may be the result of the concentration effect of the reduced endosperm and may further increase by switching carbon from starch synthesis to β-glucan synthesis.

Thus, the grain of a barley plant preferably has a β-glucan content that is higher than 6% of the total mass of unshelled grain, or more preferably higher than 7%, and most preferably higher than 8%, however, the levels of β-glucan in the waxy mutant were measured up to levels from 15 to 18%, and in the present invention can be considered as high or higher levels than these.

In a second preferred form, the barley plant grain has a reduced gelatinization temperature (as measured by differential scanning calorimetry) in addition to a relatively high amylose content. According to the data presented for example barley, this reduced gelatinization temperature is not only reduced compared to starch produced by barley with a slightly higher amylose content, but also compared to starch produced by barley having starch, which has normal levels of amylose. Thus, although lower gelatinization temperatures are considered in this invention relative to the corresponding high amylose starch, gelatinization temperatures lower than those of starch with normal amylose levels can also be considered.

In addition, in genetic backgrounds tested in this way, starch is also characterized by swelling in excess of heated water, which is weaker than the swelling of other tested starches.

In a third preferred form, starch has amylose levels above 50% of the starch content, which is a level that was never previously found in unmodified starch derived from barley.

The starch of a true barley plant has a high relative amylose content, higher than would be expected for a mutation in the SSII gene or other starch synthase gene. Thus, in wheat SSII mutants result in relative amylose levels of approximately 35% of starch. The amylose content of starch can be considered elevated when this content is significantly higher than 25%, or which is present in normal barley grain, and thus can be higher than about 30% w / w of total starch. Known barley plants, which are considered highly amylose, have a content of 35-45%. However, the present invention provides barley with an amylose content that is higher than 50%, which is a level that was never previously found in unmodified starch derived from barley.

The relative amylose content may be above 60% and, more preferably, even above 70%. It may be desirable to have even higher levels and, therefore, it was possible to achieve even higher levels in other plants by crossing with single mutations, with such levels reaching 90%. Therefore, the invention may encompass amylose levels that are above 80% or above 90%.

In a fourth preferred form, starch also has a modified structure, which leads to resistant starch. This may result from a high amylose content. Resistant starch can also form because β-glucan is present at elevated levels and is likely to have protective effects due to the binding of β-glucan to the starch granule, and the contact strength potentially provides a protective effect for starch, thereby providing stability that can be described as RS1 form, which is somewhat inaccessible to enzymatic hydrolysis. Similarly, the presence of a starch-lipid bond, as measured based on V-complex crystallinity, is also likely to contribute to the level of resistant starch. In this case, resistance is apparently the result of the physical inaccessibility of starch due to the presence of lipid, and, accordingly, it can be considered as RS1 starch. It is known that retrograded starch, which assumes a V-complex configuration, is highly resistant to enzymatic hydrolysis, and accordingly, amylopectin, which forms part of the V-complex crystalline structure, is also expected to be resistant to enzymatic hydrolysis. The starch of an example barley plant may be resistant to enzymatic hydrolysis due to the structure of the starch granule and, accordingly, may have RS2 starch. Each of these characteristics may be present separately, or in the form of two or more than two of these characteristics in combination.

Elevated levels of dietary fiber may, at least in part, take the form of resistant starch, which can be characterized by a high amylose content of starch granules, as explained above.

The relative amylose content may be above 60% and more preferably above 70%. It may be desirable to have even higher levels, and therefore it was possible to achieve even higher levels in other plants by crossing with single mutations, and such levels reach 90%. Thus, the invention may encompass amylose levels above 80% or above 90%.

It may be desirable for the barley plant to further express an altered level of activity of one or more of the synthesis enzymes of amylose or other enzymes to further increase the relative level of amylose. Thus, this barley plant may carry another mutation that further reduces or alters the biosynthesis of amylopectin, or a mutation or genetic background that enhances the biosynthesis of amylose. For example, this barley plant may exhibit an amylose extender genotype, such as a barley plant carrying the amo 1 mutation. An example of such a plant is a variety known as AC38 (also known as High Amylose Glacier).

It should be understood that the relative level of amylose discussed is a level relative to the total starch content, and therefore the rest of the starch can be predominantly an intermediate type of starch, or it can be predominantly amylopectin, or a mixture of both types. In the analyzed barley, an increased level of amylose is the result of lower levels of amylopectin, and, accordingly, the relative level of amylose is not the result of increased synthesis of amylose.

It is known that β-glucan has the effect of slowing down the enzymatic hydrolysis in the small intestine simply as a result of its presence together with another food component. Similarly, it is known that stable molecules, which have a close neighboring position with starch granules, help mask starch and contribute to its stability, making it physically inaccessible. Elevated levels of amylose and other forms of starch, which may result from a bond with a lipid, will therefore be further increased as a result of the presence and physical proximity to starch granules. Therefore, a significant increase in the effects of resistant starch is provided, as well as the provision of other beneficial effects resulting from high levels of β-glucan.

In addition, it is known that there is a dose response regarding the beneficial effects of resistant starch and β-glucan. Therefore, it is suggested that elevated levels of β-glucan along with elevated levels of resistant starch will provide enhanced health benefits.

The combination of the levels of β-glucan and resistant starch of at least the preferred variants of the present invention was not found previously and, of course, from no source without some degree of modification or purification, and therefore the forms of the present invention provide the only practical source of this benefit.

Another preferred aspect of starch is that, despite having a high relative amylose content, it also has a low gelation temperature, as measured by differential scanning calorimetry. This contradicts generally accepted information regarding the fact that high amylose starches tend to have an increased gelation temperature, which imposes limitations on the way in which high amylose starches can be used. Based on the data presented for example barley, this reduced gelatinization temperature is not only reduced compared to starch produced by lines with a slightly higher amylose content, but also compared to starch produced by barley having starch, which has normal levels of amylose. Thus, although in a preferred aspect of the present invention, reduced gelatinization temperatures are considered relative to the corresponding high amylose starch, it is also possible to consider a gelatinization temperature lower than that for starch with normal amylose levels. For high amylose starches, processing aspects that require high temperatures, therefore, inevitably require high energy consumption, which is expensive and can disrupt the functionality of other food components. Similarly, from a direct consumer perspective, high amylose starch foods may be less convenient due to the high temperature or longer cooking time. Therefore, for example, in this preferred aspect of the invention, it is now possible to offer a product, such as a noodle product, requiring the addition of boiling or heated water in a vessel, such as a cup, and not requiring heating for a long period of time, and at the same time providing the delivery of resistant starches and other nutritional components to the large intestine.

The main effect of the low gelatinization temperatures of these starches is lower temperature requirements and, therefore, the grinding energy of this food. The consequence is also that if, as may typically occur with some food processing, mixing occurs at room temperature and then this mixture is heated, a lower gelatinization temperature also reduces the time required to achieve gelation. In addition, at a temperature range below the full gelatinization temperature of normal starch, more complete gelatinization of the starch of the present invention than normal starch will occur.

One measure of gelling ability is reflected in thermal properties based on a measurement using DSC (differential scanning calorimetry). The start of the first peak (gelatinization peak) of the DSC can be at a temperature of less than 53 ° C, more preferably at a temperature of less than 50 ° C, and most preferably at a temperature of less than about 47 ° C. The beginning of the first peak can be considered as the beginning of gelatinization. Starch derived from barley grain may have a first peak at a temperature of less than about 60 ° C, more preferably at a temperature of less than 55 ° C, and most preferably at a temperature of less than 52 ° C. The ΔH (Enthalpy) of the first peak may be less than about 3.5, more preferably less than about 1.0, and most preferably less than about 0.5.

Another discovery regarding the gelatinization of flours containing starches of the present invention is that they exhibit reduced swelling. The swelling volume is typically measured by mixing starch or flour with excess water and heating to elevated temperatures, typically above 90 ° C. Then the sample is collected by centrifugation, and the swelling volume is expressed as the mass of the deposited material, divided by the dry mass of the sample. The volumes of flour swelling from waxy starches and normal barley were found to be higher than about 5.5. The swelling volumes of flour obtained from grain, which is a high amylose grain (AC38), are approximately 3.75. Whereas from the grains of the studied mutants and hybrids they are less than 3.2, preferably less than 3.0, but usually higher than about 2.

This low swelling gelatinization characteristic is especially useful if it is desired to increase the starch content of a food preparation, in particular a hydrated food preparation. In the present case, it may be desirable to increase the dietary fiber content of a sol or other liquid preparation, where otherwise there would be a restriction on the delivery of this food preparation.

This characteristic, combined with the reduced gelatinization temperature exhibited by real starch, provides the prospect of a significant increase in the nutritional benefits of foods where there is a need for quick cooking, such as instant soups and instant noodles.

It has been postulated that the effects of gelatinization temperature are the result of the altered structure of amylopectin in the grain endosperm, and one of the measurements of this structure is the distribution of chain lengths (degrees of polymerization) of starch molecules after removal of the chain branching by isoamylase. An analysis of the chain lengths of the amylopectin content of starch of SSII mutants, which serve as an example, showed that when branching is removed, they have a chain length distribution in the range of 5 to 60, which is shorter than the starch distribution that gives non-mutant lines after chain branching is removed. Starch with shorter chain lengths will also have a commensurate increase in branching frequency. Thus, this starch may also have a distribution of shorter chain lengths of amylopectin. The proportion of starch chains that have a degree of polymerization that falls within the range of 6 to 11 residues may be more than 25%, more preferably more than 30%, and most preferably more than 35%. The proportion of starch chains that have a degree of polymerization that falls within the range of 12-30 residues may be less than 65%, more preferably less than 60%, and most preferably less than about 55%. The proportion of starch chains that have a degree of polymerization that falls within the range of 31-60 residues may be less than 10%, more preferably less than 8%, but also preferably more than about 5%, and most preferably more than about 6%. Rather than taken individually, combinations of the proportions of the three ranges of chain lengths can be considered as an indicator that the starch is starch of the type that corresponds to the present invention.

A decrease in the distribution of chain lengths probably contributes to low gelation temperatures. It is also believed that the reduced chain length improves the organoleptic properties of starch, in particular, the taste sensation, thereby possibly contributing to the uniformity of the product. In addition, it has been postulated that a reduced chain length of amylopectin can reduce the degree of destruction of amylopectin, which affects the quality of food, for example, it is considered important when staling bread.

In addition, it was shown that the starch structure in starch, which serves as an example, is characterized in that the degree of crystallinity is reduced in comparison with normal starch isolated from barley. When combined with a reduced distribution of amylopectin chain lengths, reduced granular crystallinity may indicate that the gelatinization temperature will be lower. It is also believed that the reduced crystallinity of starch is associated with improved organoleptic properties and, like the shorter chain length of amylopectin, contributes to a more uniform taste experience. Thus, starch can further exhibit reduced crystallinity as a result of reduced activity levels of one or more of the amylopectin synthesis enzyme. The proportion of starch exhibiting crystallinity may be less than about 20%, and preferably less than about 15%.

An additional measure of the properties of real starch is the measurement of viscosity. Using Rapid Visco Analyzer (Rapid Viscosity Analyzer), it was found that the peak viscosity of the starch of this invention is significantly different from that of normal and waxy starches and high amylose starches derived from barley. These measurements were carried out on whole grain flour, however, the properties of starch in these measurements will prevail. Normal and waxy starches have a peak viscosity of about 900 to about 500 RVA units, the known high amylose starch has a peak viscosity of more than 200, while the starch of barley plants of the present invention has a peak viscosity of less than 100, with most less than about 50, in some plants as low as about 10 units of RVA. One skilled in the art should understand that the parameters mentioned are empirical units, and the results mentioned are intended to indicate the relative characteristics of these starches in RVA devices or similar devices, such as an amylograph.

In addition to the reduced crystallinity explained above, true starch can be characterized by the presence of a V-complex form of starch. The inventors believe that for the first time this particular form of starch was demonstrated in significant quantities in starch granules of cereal. This form of starch is usually associated with retrograde starch, in particular if it is in contact with lipids. In the case of the present invention, it has been postulated that the structure of starch allows a close relationship between plant lipids and starch to be formed, which results in a V-complex structure. It is believed that this form of starch may have health benefits, since it has reduced digestibility and, therefore, can contribute to resistant starch.

Other forms of structure may also result from lipid-starch interactions and include non-crystalline lipid-starch complexes. Thus, it can also be said that the present invention relates to a barley plant, showing appreciable amounts of starch-lipid complexes in the starch content of its grain endosperm, resulting from reduced levels of activity of one or more of the amylopectin synthesis enzyme. Starches that contain starch-lipid complexes, including those that exhibit a V-complex structure, are also usually resistant to enzymatic hydrolysis and therefore contribute to dietary fiber levels. Preferably, the proportion of crystalline starch exhibiting a crystallinity form characteristic of the starch-lipid complex is higher than about 50%, and more preferably higher than about 80%.

Starch, in addition to the presence of a V-complex form of starch, may also not show appreciable amounts of A-complex forms of starch. The absence of the A complex can be considered as an indicator of the presence of starch according to this invention.

It was also found that the temperature of pasteurization of starches and product obtained from grain according to this invention is significantly increased. The pasting temperatures of known starches are less than 70 ° C. for both normal and high amylose starches. The starches of the present invention, however, preferably exhibit pasteurization temperatures higher than about 75 ° C, or more preferably higher than about 80 ° C. It should be noted that these characteristics are empirical, and can be considered as related to such a measurement of other starches.

It has been found that the starch of the barley plant serving as an example has significant amounts of dietary fiber and resistant starch, and this increase is mainly, at least in part, the result of a high relative level of amylose, however, the contribution to dietary fiber may also be the result of starch / lipid complexes including the V complex, or a close bond of amylose or amylopectin with β-glucan. Similarly, simply elevated levels of β-glucan can also contribute significantly to increased levels of dietary fiber.

The increased relative amylose levels in the endosperm of the barley plant, which serves as an example, are likely to be the result of altered amylopectin production as a result of a decrease in the level of SSII enzyme activity.

Mutations in the gene encoding this enzyme can be expected to show up in increased amylose and / or in a decrease in amylopectin. In cases where only amylopectin synthesis is reduced, starch exhibits an increased relative level of amylose.

The reduced activity of the amylopectin synthesis enzyme can be achieved by suitable mutations within the corresponding gene or regulatory sequences of that gene. The degree to which this gene is inhibited will to some extent determine the characteristics of the resulting starch. The mutations that serve as an example according to the invention, which are SSII barley mutations, are truncation mutants, and it is known that they have a significant effect on the nature of starch, however, the altered structure of amylopectin will also be the result of a mutant with a seeping parent phenotype that sufficiently reduces activity an amylopectin synthesis enzyme to provide an interesting characteristic of starch or barley grain. Other chromosomal rearrangements may also be effective, and they may include deletions, inversions, duplications, or point mutations.

Such mutations can be introduced into the desired genetic backgrounds either by mutagenesis of the varieties of interest, but, more reliably, by crossing the mutant with the plant of the desired genetic background and carrying out a suitable number of backcrosses to displace the initially undesirable parental genetic background. Isolation of mutations can be achieved by selection of plants subjected to mutagenesis.

As an alternative to conventional methods, a molecular biological approach can be taken. The sequence of SSII is presented in this description. Vectors carrying the desired mutation and a selectable marker can be introduced into plant tissue cultures or into suitable plant systems such as protoplasts. Plants where the mutation is integrated into the chromosome to replace the existing wild-type allele can be selected using, for example, the use of a suitable probe, which is a nucleic acid specific for this mutation, and observing the phenotype. Methods for transforming monocotyledonous plants such as barley and regenerating plants from protoplasts or immature plant embryos are well known in the art, see, for example, Canadian Patent Application 2092588 Nehra, Australian Patent Application No. 61,781 / 94 National Research Council of Canada, Australian Patent No. 666939 Japan Tobacco Inc., International Patent Application PCT / US97 / 10621 by Monsanto Company, US Patent 5,589,617, and other methods are set forth in WO99 / 14314.

You can also take other well-known approaches to changing the activity of the enzyme synthesis of amylopectin, other than the use of mutations. For example, this can be done by expressing suitable antisense molecules that interfere with the transcription or processing of the gene or genes encoding the amylopectin synthesis enzyme. They can be based on the DNA sequence described here for the barley SSII gene. These can be antisense sequences for structural genes or for sequences that control the expression of a gene or splicing event. These sequences are referenced above. Methods for developing antisense sequences are well known in the art, and examples thereof can be found, for example, in US Pat. implementation of antisense techniques. Methods for introducing and maintaining such sequences in plants are also published and known.

A variant of the antisense technique is the use of ribozymes. Ribozymes are RNA molecules with an enzymatic function that can cleave other RNA molecules at specific sites defined by an antisense sequence. RNA cleavage blocks the expression of the target gene. Reference is made to EP 0321201 and WO 97/45545.

Another molecular biological approach that can also be used is cosuppression. The mechanism of cosuppression is not fully understood, but it involves the introduction of an extra copy of the gene in a normal orientation into the plant. In some cases, this additional copy of the gene interferes with the expression of the target gene of the plant. Methods for implementing cosuppression technology are referenced in WO 97/20936 and EP 0465572.

The next method that can be used using DNA sequences is gene suppression mediated by duplex or double-stranded RNA. This method uses DNA, which directs the synthesis of a double-stranded RNA product. The presence of this double-stranded molecule triggers a response from the plant’s defense system, which destroys both double-stranded RNA and also RNA from the target gene of the plant, effectively reducing or eliminating the activity of this target gene. Methods of applying this technique are referenced in Australian patent 99/292514-A and WO 99/53050.

It should be understood that this invention is likely the result of lowering the activity levels of two or more than two of the above genes using the molecular biological approach.

One of the important products, which can be considered, in particular, as a result of a high content of amylose and a high content of β-glucan, is a low-calorie product with a low glycemic index. A low-calorie product can be based on the inclusion of flour obtained from crushed grain. However, it may be desirable to first process this grain into pearl barley, removing perhaps 10% or 20% of the mass / mass of grain, thereby removing the aleurone layer, and also removing the nucleus with larger grinding. The effect of the stage of processing barley into pearl barley is to reduce the lipid content and, therefore, to reduce the calorie content of the food product. Such foods will have the effect of saturating, improving the health of the large intestine, lowering the serum concentration of glucose and lipid after eating, and also providing a low-calorie food product. The use of the product in the form of pearl barley will result in a decrease in the nutritional value provided by the aleurone layer and the germ. Flour obtained from the product in the form of pearl barley probably has an improved appearance, since the product obtained in this way has a tendency to white.

Aspects of the present invention are also the result of a combination of the aleurone layer and the germ in combination with high levels of dietary fiber. Specifically, this is the result of several higher relative levels of an aleuron or germ present in the example grain. First, barley has a significantly higher aleurone layer than other commercially available cereals, as a result of the presence of a three-cell aleurone layer. Secondly, the barley grain, which is an example, is also shriveled, which means that the endosperm is present in reduced amounts, resulting in the fact that the aleurone layer and the germ are present in increased relative amounts. Thus, barley has a relatively high level of certain beneficial elements or vitamins in combination with a sustainable starch delivery system, and such elements include divalent cations, such as bioavailable Ca ⁺⁺ , and vitamins, such as folate, or antioxidants, such as tocopherols and tocotrienols. Thus, it was found that calcium provides material for the growth and formation of bone and other calcified tissue and reduces the risk of osteoporosis in later periods of life. Folic acid has been found to have a protective effect against neural tube defects when used wisely and reduces the risk of cardiovascular disease, thereby enhancing the effects of the combination of resistant starch and β-glucan. Folic acid is also believed to have the effect of reducing the risk of certain types of cancer. Tocopherol and tocotrienols are beneficial as antioxidants, and are believed to reduce the risk of cancer and heart disease, and they have the effect of reducing the undesirable effects of oxidizing food components such as fatty acids, which can lead to rancidity. While these components of this preferred form of barley grain or the products obtained from them are conveniently placed in one grain. One of the specific forms of the ground product may be one where the aleurone layer is included in the ground product. A specific grinding process can be carried out so as to increase the amount of the aleurone layer in the crushed product. This method is referred to by Fenech et al. ((1999), J. Nutr, 129: 1114-1119). Thus, any product obtained from grain, crushed or otherwise processed to include the aleurone layer and the germ, will have additional nutritional benefits without requiring the addition of these elements from separate sources.

It should be understood that the barley plant of the present invention is preferably a plant having a grain, which is useful for obtaining food products and, in particular, for the industrial production of food products. Such preparation may include the production of flour or another product, which may be an ingredient in industrial food production. A lower level of suitability may be a starch content higher than about 12%, or possibly higher than about 15%. Or, in a similar way, it may include the ability to grind grain; thus, although barley in the form of pearl barley can be produced from most forms of grain, some grain configurations are particularly resistant to grinding. Another characteristic that may affect the variety of products of industrially applicable grain is the discoloration of the produced product. Thus, if the husk or other part of the grain shows a significant coloring, for example purple, it will manifest itself through the entire product and limit its industrial applications only to applications such as the bread component containing colored whole or crushed grains. As a rule, it is also more convenient for barley plants to be bloodless, since the presence of husks on barley grains introduces considerable difficulties in the processing of grain. Another aspect that can give a barley plant a higher value is an aspect based on the extraction of starch from grain, with higher degrees of extraction being more beneficial. The shape of the grain is also another sign that can affect the industrial suitability of the plant, so the shape of the grain can affect the lightness or something else that can be used to grind it, for example, the grain of the barley plant MK6827 has an unusually high oblong grain morphology, which complicates its grinding and processing. A convenient characteristic of this oblong shape and applicability is the ratio of two morphological characteristics - grain length to grain thickness (L / T ratio). This relationship is often due to the nature of starch. The inventors found that MK6827 has an L / T ratio greater than 6. Barley plants subjected to such selection, carrying the mutant SSII gene, have an L / T ratio ranging from about 4 to about 5, although it is expected that it can even be expanded to a larger range, so that they are still useful, possibly less than about 5.8 or at least 5.5.

The desired genetic background will include considerations of industrial yield and other characteristics. Such characteristics may include whether it is desirable to have winter or spring type of barley, an agronomic characteristic, disease resistance and resistance to abiotic stress. In Australia, it may be desirable to cross-breed with barley varieties such as Sloop, Schooner, Chebec, Franklin, Arapiles, Tantangara, Galleon, Gairdner or Picolla. The examples presented are specific to the Australian Industrial Area, and other varieties may be suitable for other growing areas.

A more complete grain may be desirable with respect to achieving higher yields and some of the advantages that can be achieved by this invention, such as producing starch with high levels of amylose or a variant of starch with altered chain length distributions. Other aspects of the present invention can, however, be better achieved with grain, which is less full. Thus, the proportion of the aleurone layer or germ in relation to starch can be higher in less full grain, thereby providing barley flour or another product that has more useful constituents of the aleurone layer. A product with a high aleurone layer may thus have a higher content of certain vitamins, such as folate, or a higher content of certain minerals, such as calcium, and this in combination with higher levels of resistant starch and / or higher levels of β- glucan can provide synergistic effects, such as providing enhanced absorption of minerals in the colon.

In order to maximize the amount of amylose, it may also be desirable for a barley plant to have other phenotypic characteristics in addition to the reduced activity of one or more of the amylopectin synthesis enzyme. The genetic background may therefore also include an additional high amylose phenotype, for example, the amo 1 mutation in AC38 (the causative gene is unknown) and the waxy mutation (found, for example, in the Waxiro variety). Additionally, it may be desirable to obtain double mutations in other available barley mutants with shrunken endosperms where the causative gene is unknown.

In a further aspect, the invention relates to grain obtained from a barley plant, referring to this description.

It should also be understood that the scope of the present invention includes processed grain, including chopped, ground, crushed, in the form of pearl barley or cereal flakes, or a product obtained from the processed or whole grain barley plants referred to above, including flour. These products can then be used in various food products, for example in flour products, such as bread, cakes, biscuits and the like, or in food additives, such as thickeners, or for malt or other barley drinks, noodles and instant soups.

Alternatively, starch isolated from the grain of a barley plant referred to above is included within the scope of this invention. Starch can be isolated using known techniques.

It should be understood that one of the advantages of the present invention is that it offers one or more than one product that has a particular nutritional value, and, moreover, this is done without the need to modify starch or other constituents of barley grain.

However, it may be desirable to make modifications of starch, β-glucan or other constituent of the grain, and such a modified constituent is within the scope of the invention.

Modification methods are known and include the extraction of starch or β-glucan, or other constituent parts by conventional methods, and the modification of starches to obtain the desired stable form.

Thus, starch or β-glucan can be modified either once or repeatedly by using a treatment selected from the group including, but not limited to, heating and / or moistening, physical treatment (e.g., ball mill crushing), enzymatic (when using, for example, α- or β-amylase, pullallanase, or the like), chemical hydrolysis (wet or dry using liquid or gaseous reagents), oxidation, cross-linking with difunctional reagents (for example measures, sodium trimethaphosphate, phosphorus oxychloride) or carboxymethylation.

The dietary fiber content of barley grain, which serves as an example, is not solely the result of increased relative amylose content in the endosperm. One of the main reasons is that β-glucan is present at elevated levels and makes a significant contribution to the level of dietary fiber. There are probably also protective effects due to the binding of β-glucan to the starch granule, the strength of this bond potentially providing a protective effect for starch, thereby ensuring stability, which can be described as the RS1 form, which is somewhat inaccessible to enzymatic hydrolysis. Similarly, the presence of a starch-lipid bond, as measured based on V-complex crystallinity, is also likely to contribute to the level of stable carbohydrate. In this case, resistance is likely due to physical inaccessibility due to the presence of lipid and, accordingly, it can be considered as RS1 starch. Thus, it is known that retrograde starch, which assumes a V-complex configuration, is highly resistant to enzymatic hydrolysis and, accordingly, it is expected that amylopectin, which forms part of a starch granule having a V-complex crystalline structure, will have enhanced resistance to enzymatic hydrolysis . Thirdly, the starch of the barley plant, which serves as an example, may be resistant to enzymatic hydrolysis due to the structure of the starch granule and, accordingly, may have RS2 starch.

It should be understood that, although various indications are given as aspects of the present invention, the present invention may relate to combinations of two or more than two aspects of the present invention.

EXAMPLE 1

Background

The synthesis of starch in the endosperm of higher plants is carried out by a number of enzymes that catalyze four key stages. First, ADP-glucose pyrophosphorylase activates the monomeric starch precursor by synthesizing ADP-glucose from G-1-P and ATP. Secondly, the activated glycosyl donor, ADP-glucose, is transferred to the non-reducing end of the pre-existing α-1-4 linkage of starch synthases. Thirdly, starch branching enzymes introduce branch points by cleaving a portion of α-1,4-linked glucan, followed by transferring the cleaved chain to an acceptor chain to form a new α-1,6 bond. Finally, genetic studies show that starch branching removal enzymes are essential for synthesizing normal amounts of starch in higher plants, however, the mechanism by which branching branching enzymes act is unclear (Myers et at., 2000).

Although it is clear that at least these four activities are necessary for the synthesis of normal starch granules in higher plants, multiple isoforms of each of these four activities were found in the endosperm of higher plants, and specific roles were suggested for individual isoforms based on mutational analysis (Wang et al., 1998, Buleon et al., 1998) or by modifying gene expression levels using transgenic approaches (Abel et al., 1996, Jobling et al., 1999, Scwall et al., 2000). However, the exact contributions of each isoform of each activity to starch biosynthesis are still unknown, and it is not known whether these contributions differ significantly between species. Two isoforms of ADP-glucose pyrophosphorylase, one form inside the amyloplast and one form in the cytoplasm, are present in the cereal endosperm (Denyer et al., 1996, Thorbjornsen et al., 1996). Each form consists of two types of subunits. The mutants shrunken (sh2) and brittle (bt2) of maize mean damage to the large and small subunits, respectively (Girouz and Hannah, 1994). Four classes of starch synthase were found in the cereal endosperm, an isoform exclusively localized inside the starch granule, granule-bound starch synthase (GBSS), two forms that are distributed between the granule and the soluble fraction (SSI, Li et al., 1999a SSII, Li et al., 1999b) and the fourth form, which is completely localized in the soluble fraction, SSIII (Cao et al., 2000, Li et al., 1999b, Li et al., 2000). GBSS has been shown to be essential for the synthesis of amylose (Shure et al., 1983), and it has been shown that mutations in SSII and SSIII alter the structure of amylopectin (Gao et al., 1998, Craig et al., 1998). Mutations that determine the role of SSI activity are not described.

Three forms of a branching enzyme are expressed in the cereal endosperm, branching enzyme I (BEI, branching enzyme I), branching enzyme IIa (BEIIa) and branching enzyme IIb (BEIIb) (Hedman and Boyer, 1982, Boyer and Preiss, 1978, Mizuno et al ., 1992, Sun et al., 1997). In maize and rice, high amylose phenotypes are the result of damage to the BEIIb gene (Boyer and Preiss, 1981, Mizuno et al., 1993). In these mutants, the amylose content is significantly increased, and the branching frequency of residual amylopectin is reduced. In addition, there is a significant pool of material that is defined as “intermediate” between amylose and amylopectin (Boyer et al., 1980, Takeda et al., 1993). Mutations that determine the roles of BEIIa and BEI still need to be described, despite the fact that in potatoes, negative regulation of BEI alone has minimal effect on starch structure (Filpse et al., 1996). However, in potatoes, the combination of negative regulation of BEII and BEI gives a much higher amylose content than negative regulation of BEII alone (Schwall et al., 2000). Two types of branching branching enzymes are present in higher plants and are determined based on their substrate specificity, branching branching enzymes, such as isoamylase, and branching branching enzymes, such as pullulanase (Myers et al., 2000). Sugary-1 mutations in corn and rice are associated with a deficiency in both branching enzymes (James et al., 1995, Kubo et al., 1999), however, the causative mutation is mapped to the same location as the branching enzyme gene chains, such as isoamylase. The mutant Chlamydomonas sta-7 (Mouille et al., 1996), an analogue of the corn sugary-1 mutation, has a negative regulation of one isoamylase activity.

The known variation in the structure of barley starch is limited compared to the variation available in corn. Most of the highly characterized mutations are the waxy and high amylose mutations identified as AC38. Binary mutants are also designed and analyzed (Schondelmaier et al., 1992, Fujita et al., 1999). A wide range of characteristics of varying the structure and properties of starch is reported (Czuchajowska et al., 1992; Schondelmaier et al., 1992; Vasanthan and Bhatty, 1995; Morrison et al., 1984; Gerring and DeHaas, 1974; Bankes et al., 1971 ; Persson and Christerson, 1997; Vasanthan and Bhatty, 1998; Czuchajowska et al., 1998; Song and Jane, 2000; Andreev et al., 1999; Yoshimoto et al., 2000), as well as grain properties (Swantson, 1992, Ahokas, 1979; Oscarsson et al., 1997; Oscarsson et al., 1998; Andersson et al., 1999; Elfverson et al., 1999; Bhatty, 1999; Zheng et al., 2000; Izydorczyk et al., 2000; Andersson et al., 2000), and the utility of these mutants in animal feeding experiments has been investigated (Xue et al., 1996; Newman et al., 1978; Calvert et al., 1976; Wilson et al., 1975; Sundberg et al. , 1998; Bergh et al., 1999), food products human learning (Swantson et al., 1995; Fastnaught et al., 1996; Persson et al., 1996; Pomeranz et al., 1972) and human nutrition (Pomeranz 1992; Granfeldt et al., 1994; Oscarsson et al., 1996; Akerberg et al., 1998).

In the present example, the inventors isolated a new class of high amylose mutant from barley. These mutant lines have an amylose content (65-70%), higher than that of the well characterized High Amylose Glacier (AC38) mutant (45-48%) (Walker et al., 1968), and have starch with an amylopectin structure in which there is an increase in the branching frequency of starch, which is the opposite of the reduced branching frequency associated with the amylose extender mutant in maize (Takeda et al., 1993).

The characteristics of the grain and starch of this mutant are investigated in detail, and the causal mutation is mapped. The isolated mutations are allelic to the previously known shrunken mutation in barley, sex6, and it was shown that the causative mutation is localized inside the starch synthase gene II. The effects of this mutation shed new light on the starch biosynthesis process and illustrate how mutations in specific genes can have different effects on the structure of starch from species to species.

Materials and methods

Mutagenesis and selection

The Himalaya barley variety was mutagenized using sodium azide according to Zwar and Chandler (1995). Selection of variants with altered grain morphology was performed according to Green et al. (1997). Only 75 lines with shrunken endosperm phenotypes were identified and maintained according to Green et al. (1997).

Starch Isolation

Starch was isolated from barley grain using the method of Schulman et al. (1991).

Amylose determination methods

The determination of the amylose / amylopectin ratio by HPLC for the separation of unbranched chain starches and by the iodine binding method was carried out as described by Batey and Curtin (1996). Analysis of the amylose / amylopectin ratio by analysis of branched chain starches was carried out according to Case et al. (1998).

Starch Measurement

Starch was determined using the total starch analysis kit supplied by Megazyme (Bray, Co Wicklow, Republic of Ireland).

Protein content

Nitrogen was determined by the Kjeldahl method, and protein content was calculated using factor 5.7.

Β-glucan levels

β-Glucan was determined using a kit supplied by Megazyme (Bray, Co. Wicklow, Republic of Ireland).

Starch chain length distribution

Starches were removed by branching, and chain length distributions were analyzed using carbohydrate electrophoresis using fluorophore (FACE, fluorophore assisted carbohydrate electrophoresis) using capillary electrophoresis according to Morell et al. (1998).

DSC

Gelatinization was measured in a Pyris 1 differential scanning calorimeter (Perkin Elmer, Norwalk CT, USA). Starch was mixed with water in a ratio of 2 parts of water: 1 part of starch, and this mixture (40-50 mg, accurately weighed) was placed in a stainless steel bowl and tightly closed. The sample was scanned at 10 ° C for a minute from 20 ° C to 140 ° C with an empty stainless steel bowl as a comparison. Gelatinization temperatures and enthalpy were determined using Pyris software.

RVA analysis

Viscosity was measured on a Rapid-Visco-Analyzer (RVA, Newport Scientific Pty Ltd, Warriewood, Sydney) using conditions as described by Batey et al., 1997, for wholemeal. To inhibit α-amylases, silver nitrate at a concentration of 12 mM was included in all assays. The measured parameters were peak viscosity (maximum viscosity of hot paste), retention force, final viscosity and paste temperature. In addition, decomposition (peak viscosity minus retention force) and delay (final viscosity minus retention force) were calculated.

Flour swelling

The amount of flour swelling was determined according to the method of Konik-Rose et al. (2001).

X-ray analysis data

X-ray diffraction data was collected using standard techniques (Buleon et al., 1998).

Scanning Electron Microscopy

Scanning electron microscopy was performed using Joel JSM 35C equipment. Purified starches were sprayed with gold and scanned at 15 kV at room temperature.

Getting double haploids

Double haploids were obtained from F1 plants, originating from crosses between 292 and Hordeum vulgare cv Tantangara and between 342 and H.vulgare cv Tantangara Dr. P. Davies, Waite Institute, Adelaide, Australia.

Clutch analysis

Genetic linkage data was calculated using MapManager.

Construction of the barley cDNA library

Five mg of polyA + mRNA from barley endosperm tissues on the 10th, 12th and 15th day after pollination was used for cDNA synthesis according to the protocols (Life Technology). Notl- (dT) 18 primer (Pharmacia Biotech) was used to synthesize the first cDNA strand. Double-stranded cDNAs were ligated with a Sa / I-XhoI adapter (Stratagene) and cloned to the shoulders of a Sa / I-NotI ZipLox (Life Technology) after enzymatic hydrolysis of the cDNA using NotI followed by size fractionation (SizeStep 400 spun column from Pharmacia Biotech). Ligated cDNAs were packaged with Gigapack III Gold packaging extract (Stratagene). The library titer tested by E. coli strain Y1090 (ZL) was 2 × 10 ⁶ b.p. (plaque forming units).

Cloning of specific sections of starch II starch synthase cDNA using PCR

The wSSIIp1 cDNA clone was used to screen the barley cDNA library. This wSSIIp1 cDNA clone was obtained by PCR using the primers ssIIa (TGTTGAGGTTCCATGGCACGTTC SEQ ID NO 9) and ssIIb (AGTCGTTCTGCCGTATGATGTCG SEQ ID NO 10), amplifying the region between the positions of the 141 and 2135 nucleotides AF1573535B of Gen 1835 and 1835 of the Generic Genetic Principle of Genesis 1135.

Amplification was carried out using an FTS-1 thermal sequencer (Corbett, Australia) for 1 cycle at 95 ° C for 2 minutes; 35 cycles at 95 ° C for 30 seconds, at 60 ° C for 1 minute, at 72 ° C for 2 minutes and 1 cycle at 25 ° C for 1 minute. The wSSIIp1 fragment was cloned into the pGEM-T vector (Promega).

Barley cDNA library screening

A cDNA library constructed from RNA from the barley endosperm cv Himalaya was screened by a 347 bp cDNA fragment, wSSIIp1, under the hybridization conditions described previously (Rahman et al., 1998). Hybridization was carried out in 50% formamide, 6x SSPE, 0.5% SDS, 5x Denhardt's solution and 1.7 μg / ml salmon sperm DNA at 42 ° C for 16 h, then washed with 3x 2x SSC containing 0.1% SDS , at 65 ° C for 1 hour for washing.

Barley Genomic Library Screening

A barley genomic library (cv Morex barley) was constructed and screened essentially as described in Gubler et al (2000) using barley SSII cDNA as a probe.

Genomic clone sequencing

The SSII Morex gene was subcloned into plasmid vectors and sequenced. Genes 292 and MK6827 were sequenced using PCR amplification of the overlapping regions of this gene using primers designed based on the Morex sequence. PCR fragments were either sequenced directly or subcloned and sequenced from plasmids.

Identification of expressed sites

The regions of genomic sequences 292 and MK6827, presumably present in the cDNA, were determined in comparison with the Himalaya cDNA sequence and the Morex genomic sequence.

PCR analysis of G to A mutations in the SSII gene

PCR primers were designed that amplify the region containing the G to A transition identified in 292. The primer sequences are: ZLSS2P4 (CCTGGAACACTTCAGACTGTACG SEQ ID NO 11) and ZLBSSII5 (CTTCAGGGAGAAGTTGGTGTAGCQQQ. Amplification was performed using an FTS-1 thermal sequencer (Corbett, Australia) for 1 cycle at 95 ° C for 2 minutes; 35 cycles at 95 ° C for 30 seconds, at 60 ° C for 1 minute, at 72 ° C for 2 minutes and 1 cycle at 25 ° C for 1 minute.

Analysis of barley endosperm proteins in SDS-PAGE electrophoresis

Starch was obtained from developing and mature endosperm of barley and wheat, and surface proteins were removed using proteinase K, as described (Rahman et al., 1995). Proteins of starch granules were extracted from 20 mg of starch (dry weight) using 0.5 ml of extraction buffer containing 50 mm Tris pH 6.8, 10% SDS and 10% 2-mercaptoethanol. After gelatinization by boiling for 10 min and collecting the starch by centrifugation, 15 microliters of the supernatant was applied to each lane.

Getting double haploids

Double haploids were obtained from F1 plants, originating from crosses between 292 and Hordeum vulgare cv Tantangara and between 342 and H.vulgare cv Tantangara Dr. P. Davies, Waite Institute, Adelaide, Australia.

Backcrossing strategy

Crosses were made between 292 and Hordeum vutgare cv Sloop to obtain seed F1. Plants grown from seed F1 were self-pollinated to produce a seed population of F2. Plants grown from this seed F2 were tested using PCR analysis, and plants homozygous for mutation 292 were backcrossed with Sloop (BC1). Plants F1 resulting from BC1 were again tested by PCR, and plants heterozygous for mutation 292 were selected and backcrossed with Sloop (BC2). Plants F1 obtained as a result of BC2 were again analyzed by PCR and plants heterozygous for mutation 292 were selected. These plants were either self-pollinated to obtain a BC2F2 population or cross-bred again with Sloop (BC3). Plants F1 resulting from BC3 were again analyzed by PCR and plants heterozygous for mutation 292 were selected. These plants were self-pollinated to obtain a BC3F2 population. Plants grown from this seed were tested by PCR, and plants homozygous for mutation 292 were selected to generate a single seed generation and seed propagation.

results

Mutant selection

The identification of a number of mutants in the Himalaya barley variety without husk or with seedless seeds induced by sodium azide treatment was previously reported by Zwar and Chandler (1995). A group of 75 shriveled grain mutants was identified by the inventors, and the amylose starch content of this shriveled seed was determined by HPLC (Figure 1). It was found that two lines, 292 and 342, had an amylose content of 71 and 62.5%, respectively (Table 1). The content of amylose 292 and 342 was significantly higher than in the AC38 line, previously well characterized (47% amylose, see Table 1). In this study, the genetic basis of a new high amylose phenotype exhibited by 292 and 342 was determined and the effects of causative mutation on the structure and functionality of grain and starch were described.

Grain characteristics

Grain size and morphology

The effects of the mutation on the mass and morphology of the grain are noted (Table 2). Grain mass reduced from 51 mg for the Himalaya parent line to 32 mg for 292 and 35 mg for 342. These mutants retained the length and width as in the wild type, but in comparison the grains are flattened (from 2.82 mm of the average thickness in Himalaya to 1.58 and 1.75 mm at 292 and 342, respectively) and have a substantially unfilled central portion. Figure 2 shows photographs of grain mutant and wild type. Grain sizes were measured in the traditional way, grain length (L), grain width at the widest point (W) and thickness (T), as indicated in FIG. 2. The ratio of length (L) to thickness (T) is a useful diagnostic feature for this mutation at values> 3.5, typically found for seeds bearing mutations 292 or 342, and values <3.5 for non-mutant barley plants.

Grain composition:

The starch content of the mutant lines was reduced from 49.0% for Himalaya to 17.7 and 21.9% for 292 and 342, respectively (see Table 1). Subtraction of the mass of starch from the total mass of grain to obtain the total non-starch content of grain showed that the loss of starch content occurs due to the loss of mass of grain at masses of non-starch contents of 26.0, 26.3 and 27.3 mg for Himalaya, 292 and 342, respectively.

The protein content of 292 and 342 is increased relative to the parental Himalaya line (Table 1), however, this effect occurs due to the loss of starch from grain and is not associated with any increase in protein synthesis per grain.

The levels of β-glucan of mutants 292 and 342 are also elevated and are higher than would be expected as a result of the effect of decreasing starch content (Table 1). In both cases, the content of β-glucan is increased by about 20% per caryopsis, which, possibly, is a switch of a small part of the incoming carbon from starch synthesis to β-glucan synthesis.

The composition and functionality of starch

Amylose and Amylopectin

The amylose content was determined using two methods, firstly, displacing by size HPLC in 90% (v / v) DMSO and, secondly, the indicator of blue staining with iodine. The amylose content determined by each method was similar, and the HPLC data are presented in Table. one.

Based on data on grain mass and amylose content for mutant lines and wild-type lines, it is possible to calculate the amount of amylose stored per grain. This analysis shows that there is a decrease in the amount of amylose per grain from 6.2 mg / grain in Himalaya to 4.0 mg / grain in 292 and 4.8 mg / grain in 342. On the contrary, there is a sharp decrease in the synthesis of amylopectin per grain from 18 7 mg in Himalaya to 1.6 mg in 292 and 2.9 mg in 342.

Chain length distribution

The distribution of starch chain lengths after removal of the branching chain with isoamylase was carried out using carbohydrate electrophoresis using fluorophore (FACE). The distribution of the chain lengths of the mutants 292 and 342 and Himalaya is shown in Fig. 3a. Figure 3b shows a graph of differences in which the normalized distribution of chain lengths for mutants 292 and 342 is subtracted from the normalized distribution of Himalaya. Percentages of chain lengths from DP 6-11, DP 12-30, and DP 31-65 are calculated and presented in Table. 3. There is a noticeable shift in the distribution of chain lengths for mutants 292 and 342, such that in the section from DP 6-11 the percentage of chains is higher compared to DP 12-30.

Differential Scanning Calorimetry

The gelation temperature of the mutants was investigated using differential scanning calorimetry, and the data are presented in Table. 4. Both 292 and 342 produce starches that have significantly lower gelatinization temperatures than Himalaya starches with respect to the initial, peak and final temperatures for peak gelatinization. The gelatinization peak enthalpy for mutants 292 and 342 is also sharply reduced compared to the wild type. The initial temperature of the amylose / lipid peak is also reduced for mutants 292 and 342, however, the enthalpy is increased, which corresponds to an increased content of amylose in the mutants.

RVA starch viscosity

An RVA analysis of wholemeal barley flour samples was performed to examine their pasting viscosity. Previous studies have shown that analysis of wholemeal samples has a strong correlation with analysis of isolated starches (Batey et al., 1997). This analysis showed that there are large differences between the investigated barley genotypes (see table. 5 and figure 4). Two varieties of barley containing wild-type starch, Himalaya and Namoi, showed typical RVA profiles in which a protruding peak of viscosity was observed followed by a decrease in viscosity to a holding force, followed by an increase in viscosity as the temperature decreased to final viscosity. As is commonly observed for barley starches, the final viscosities for wild-type starches were equivalent to peak viscosities or below them (Table 5). AC38 obtained an outstanding peak viscosity, however, due to the increased amylose content of this line, the resulting final viscosity was higher than the peak viscosity. However, 292, 342 and MK6827 received a very different profile. Other barley starches did not produce a noticeable initial increase in viscosity corresponding to peak viscosity, and therefore no index of failure could be calculated. Values for peak viscosity given in Tab. 5 for 292, 342 and MK6827, were viscosities recorded at the peak viscosity time point for Himalaya. In 292, 342, and MK6827, viscosity increased throughout the analysis to achieve a final viscosity comparable to other wholemeal samples. When normalized based on the starch content, starches 292 and 342 had very high final viscosities (see Table 5).

The volume of swelling is a method of measuring the properties of flour and starch, with the help of which they study the behavior of this material when exposed to heat and excess water. Increased water absorption is measured by weighing the sample before mixing and after mixing this sample with water at certain temperatures and then collecting the gelled material. This analysis showed that control samples, Himalaya and Tantangara, swell 6–8 times their dry weight; on the contrary, 292 and 342 swell only up to 2-3 times their dry weight (Table 9).

Crystallinity

The structure of the starches was further investigated using x-ray crystallography (see Table 6 and Figure 5). Himalaya showed the expected pattern for cereal starch having a predominantly “A” type crystallinity, and both AC38 and Waxiro showed very similar X-ray diffraction patterns, although crystallinity levels were lower for AC38 and higher for Waxiro. For mutants 292 and 342, the X-ray diffraction pattern was shifted to a mixture of V and B patterns. In addition to a shift in the diffraction pattern, the amount of crystallinity was sharply reduced in mutants 292 and 342 to 9 and 12%, respectively. This result corresponds to the low amylopectin content of starches 292 and 342.

Pellet morphology

The starch granule morphology was investigated using scanning electron microscopy (Figure 6). The size and shape of the pellets from Himalaya (Figure 6, panel a), waxy barley (Waxiro, Figure 6, panel b) and AC38 (Figure 6, panel c) were consistent with previously published observations of starch granules in normal barley lines. The morphology of the "A" type starch granules in the mutant lines 292 (Fig. 6, panel d), 342 (Fig. 6, panel d) and MK6827 (Fig. 6, panel e) is clearly changed, since the granules have a curved surface in comparison with a smooth biconvex form of granules of normal barley.

Dietary fiber

Analysis of dietary fiber was carried out according to the AOAC method and showed that an increase in dietary fiber was observed in 292 and 342, and that such an increase in dietary fiber was more likely a result of an increase in insoluble dietary fiber than soluble dietary fiber (Table 1), in accordance with the components of dietary fiber fiber, which is resistant starch and β-glucan. It should be noted that this measure of dietary fiber is a chemically determined measure, which is completely different from the physiological measure that is relevant in terms of nutrition.

Genetic basis of mutation

Segregation relation

Crossing the mutation with barley cultivars that do not exhibit the phenotype of shriveled endosperm 292 or 342 showed that this mutation is a direct recessive mutation showing a ratio of 3 normal: 1 shriveled seed F2 of outcrossing populations and a ratio of 1 normal: 1 shriveled seed of a double haploid population, developed after one outcrossing (see Table 6). A normal seed is defined as a seed with an L / T ratio <3.5, and a shriveled seed is defined as a seed with an L / T ratio> 3.5.

Allelic nature of mutants

By analyzing the offspring from crosses 292 and 342, it was shown that mutations 292 and 342 are allelic. All F1 seed, originating from reciprocal crosses, showed phenotypes of grain mass and grain morphology within the range of sizes and shapes observed for parent lines 292 and 342 and outside the range of seed size and shape found for the Himalaya parent line. In addition, all F2 seed, originating from F1 292x342 plants, exhibited a typical phenotype of shriveled seed mutants 292 and 342.

An analysis of grain morphology and starch characteristics of a series of shredded grain mutants available from the Barley Germplasm Collection (USDA-ARS, National Small Grains Germplasm Research Facility, Aberdeen, Idaho 83210, USA) suggested that the MK6827 line (BGS31, also called GSHO 2476 ), carrying the sex6 mutation, showed a number of characteristics of grain and starch, highly similar to mutations 292 and 342. Crosses were made between 292 and MK6827, and all F1 grains showed a typical phenotype 292 with respect to the phenotype of grain mass and shriveled seed. All F2 seed, originating from F1 292 × MK6827 plants, showed a phenotype of shriveled endosperm with L / T ratios> 4. In contrast, seed F2 from crossing between 292 and a commercially available Sloop barley variety gave a bimodal distribution showing a 3: 1 segregation ratio between shriveled and filled seed (Table 6). All F1 inoculum originating from crosses of 292 and 5 other lines with wrinkled endosperm phenotypes (BGS 380, wrinkled endosperm 4, 7HL (Jarvi et al., 1975); BGS 381, wrinkled endosperm 5, 7HS (Jarvi et al, 1975 ); BGS 382, sex 1, 6HL (Eslick and Ries, 1976); BGS 396, wrinkled endosperm 6, 3HL (Ramage and Eslick, 1975); BGS 397, wrinkled endosperm 7, not mapped (Ramage and Eslick, 1975), gave a grain with the morphology of the filled seed. Based on this, mutations 292, 342 and MK6827 are considered allelic, and based on previously published locations on the map for the sex6 locus, mutations 292 and 342 are probably predicted to in the short arm of the barley chromosome 7H, approximately 4 cm from the centromere (Netsvetaev, 1990, Netsvetaev and Krestinkov, 1993, Biyashev et al., 1986, Netsvetaev, 1992).

Clutch analysis

From a cross between 292 and a commercial malting barley variety, Tantangara, a double haploid population was obtained that contained 90 descendant lines (Table 8).

These lines were evaluated by seed morphology (filled against shriveled seed), chain length distribution by FACE (percentage of chains with DP 6-11), seed coat (uncoated or husked), and by PCR marker (see below). These data are presented in Table. 8. In this population, the wrinkled seed trait and FACE distribution 292 are precisely co-segregated, as might be expected if the altered grain size and shape were the result of altered starch deposition. The cosegregation of features is illustrated in FIG. Panel A shows the relationship between the starch chain length (illustrated based on the percentage of chains between DP 6-11) and the ratio of length to thickness. Empty circles indicate lines that have a PCR marker for mutation 292, crosses indicate lines that carry a wild-type PCR marker. There is a clear definition between these two groups of lines. Panel B of FIG. 8 shows the relationship between the starch chain length and the seed weight, and shows that the seed weight is less diagnostic for this mutation than the length to thickness ratio.

In barley, the presence or absence of husk is controlled by the nud locus located on chromosome 7H, and since Tantangara is husk-covered barley, and 292 is a bloodless type, this trait can be estimated in double haploid offspring. Analysis of the linkage between the sign of skinless / husked and FACE data showed that in this cross, the 292 mutation was mapped within 16.3 cm from the nud locus. This localization coincides with previous mapping data for the allelic mutation sex6 (Netsvetaev, 1990, Netsvetaev and Krestinkov, 1993, Biyashev et al., 1986, Netsvetaev, 1992).

Causative Gene Identification

It has been demonstrated that the nud gene is located on the barley chromosome 7H (Fig. 8, Fedak et al., 1972). In wheat, three starch synthases (GBSS, SSI, and SSII) and an isoamylase-type branching enzyme (S. Rahman, personal communication) are located on the short arm of chromosome 7, a homologous chromosome (Yamamori and Endo, 1996, Li et al., 1999a, Li et al., 1999b, Li et al., 2000). Close linkage with the nud locus suggested that the SSII gene is the most likely candidate gene. Wheat SSII gene was cloned at the cDNA level (Li et al., 1999b; Genbank, catalog number AF155217) and at the genomic level (Li et al., Personal communication), and barley cDNA was isolated and cloned (Fig. 9). Sequencing of the barley and wheat SSII genomic sequences showed that these genes have very similar exon / intron structures, however, the lengths of the intron regions differ between the sequences (Figure 10). Comparison of the genome sequence of Morex and the cDNA sequence from Himalaya (Figure 9) leads to the identification of the derived cDNA sequences from Morex, 292 and MK2827.

A mutation G to A mutant was found in the SSII gene of 292 at a position that corresponds to the 1829 alignment shown in FIG. 11. This mutation introduces a stop codon into the open reading frame of SSII 292 (FIG. 12). The analysis of the Tantangara and Himalaya sequences showed that both wild-type genes are identical in this region, and both 292 and 342 contain the same mutation of transition G to A. The introduced stop codon should shorten the product of this gene in such a way that full-size C - the terminal catalytic domain of the starch II synthase gene will not be translated, and therefore, there is a high probability that the activity of SSII is completely terminated by this mutation.

Transition G to A was also present in MK6827 at alignment position 242 shown in FIG. 11 and the Himalaya cDNA sequence in FIG. 9. This mutation also introduces a stop codon into the open reading frame of SSII 292 (Fig. 12) and should prevent the translation of more than 90% of the SSII gene, terminating the activity of SSII encoded by this gene.

A mutation of the G transition to A in 292 interrupted the restriction site (NIaIV) in the barley SSII gene. The localization of this NIalV diagnostic site is shown in Fig. 14, panels (a) and (b). On figv shows agarose gel electrophoresis of enzymatic hydrolysis of NIalV from barley, showing that the diagnostic picture for mutation 292 is in 292 and 342, but not in MK6827, Himalaya or Tantangara.

The PCR marker for the G to A transition was evaluated in 90 lines of a 292 × Tantangara double haploid population and found that it precisely co-aggregates with shriveled seed phenotypes and FACE chain length distribution, indicating that mutation 292 is fully linked to this starch phenotype, and that there is a high probability that this mutation is the causative mutation that underlies phenotype 292. Fig. 14g shows an analysis of lines from a double haploid population of 292 × Tantangara.

Biochemical evidence of SSII activity loss

The composition of starch biosynthesis enzymes in the mutant and normal barley lines was studied using a number of gel electrophoresis techniques. Analysis of the soluble fraction of the developing endosperm showed that all lines contained BEI, BEIIa, BEIIb, SSI and SSIII, and that the content of these isoforms BE and SS, respectively, was essentially unchanged. However, analysis of starch granules showed that several bands were absent. First, SDS-PAGE electrophoresis analysis (FIG. 16, panel B) showed that the 90 kD band that was present in Himalaya, Tantangara and AC38 was absent in 292, 342 or MK6827. Using immunoblotting, it was shown that this band contains SSII (Figure 16, panel B, as well as BEIIa and BEIIb. The discovery that BEIIa and BEIIb are present in the soluble fraction, but not in the starch granule, indicates that mutants 292, 342 and MK6827, there is more likely a change in the distribution of these enzymes than a mutation that stops expression.On the contrary, no evidence of SSII expression was found in either the soluble or granular fraction (Fig. 16, panels A and B), which coincides with genetic evidence of immediate linkage I SSII mutation to the observed phenotypes in 292, 342 and MK6827.

Selection of lines carrying the mutation 292

Two strategies have been used to transfer mutation 292 to alternative barley genotypes.

In the first example, double haploid lines from crossing between 292 and Tantangara were obtained. Data for seed coverage, seed weight, L: T ratios, chain length distributions, and SSII DNA marker status are presented in Table. 8. A more complete analysis of the composition of these lines is presented in Table. 9, including RVA analysis, β-glucan content and flour swelling volume. These data show that the lines carrying mutation 292 have significantly different RVA parameters (an example is the peak / final viscosity ratio), higher β-glucan content and altered flour swelling volumes.

In the second example, the mutation was carried out by carrying out two backcrosses of 292 with a variety having normal starch properties (cv Sloop). For analysis, seed F2 was collected from three F1 backcrossing plants 2. This seed F2 was divided into seed categories with a ratio of LT> 3.5 and a ratio of LT <3.5. The seed distribution between these classes was in line with expectations for a single recessive gene. Flour swelling data for these seed categories obtained from each plant is shown in FIG. 10 and shows that the starch swelling trait is clearly tolerated through a cross-line process with an average Sloop genetic background content of 75%.

Discussion

The inventors describe the isolation of new mutants, 292 and 342, in barley, which have a phenotype of shriveled endosperm. Analysis of the grain composition shows that the wrinkled phenotype is a consequence of a significant decrease in starch content, and analysis of the starch composition shows that this decrease is manifested as a high amylose phenotype, which is the result of a decrease in amylopectin synthesis.

Mutants 292 and 342 have a unique combination of the properties of grain and starch in the content of both elevated levels of β-glucan and stable starch. The levels of β-glucan of these lines are increased by about 15% higher than what is expected due to the effect of a reduced starch content, which suggests that carbon, unable to turn into starch, switches to the synthesis of β-glucan. Determinations of dietary fiber levels demonstrate that mutant grain has elevated levels of dietary fiber, and that this increase is a consequence of increased insoluble dietary fiber.

This combination of properties indicates that these mutants may have a very interesting potential as components of a human diet. First, elevated β-glucan levels suggest that these lines may be useful in lowering cholesterol through the well-established action of β-glucan in lowering cholesterol levels. Secondly, the presence of resistant starch indicates that these lines may be useful due to the prospect of intestinal health due to the well-established ability of resistant starches to promote fermentation in the colon (Topping et al., 1997, Topping, 1999). Thirdly, the grain composition indicates that these lines will have a low energy density and that they can undergo enzymatic hydrolysis slowly, indicating that they can contribute to the manufacture of foods with a low glycemic index.

The starch properties of the example lines are also unique in that they combine high amylose starch, which also has a low gelatinization temperature. This contradicts the high amylose mutations resulting from mutations in the branching chain enzyme IIb, in which the gelatinization temperature typically rises, as for example with the amylose extender mutation in maize (Ng et at., 1997, Katz et al., 1993, Krueger et al., 1987 , Fuwa et al., 1999). Although the amylose 292 content is comparable to the amylose extender lines, the structure of the amylopectin component of starches is dramatically different (Wang et al., 1993). For 292 and 342, the distribution of chain lengths of amylopectin is shifted towards a lower degree of polymerization, while for amylose extender, the distribution of chain lengths is shifted towards a lower degree of polymerization. This suggests that amylopectin, and not the amylose content, is the primary determining factor in the gelatinization temperature, and that this effect is mediated by the strength of the interaction between the external chains of the amylopectin molecule. Similar effects have been noted for a number of starches Jane et al., 1999.

Viscosity data based on RVA analysis indicate that starch from mutant SSII lines is markedly different from normal barley and AC38. SSII barley mutants essentially do not have a viscosity peak typically observed with a sharp increase in temperature to 95 ° C at the beginning of the RVA temperature profile. Instead, the viscosity of these mutants increases monotonously to a final viscosity, if any. These data are consistent with the low amylopectin content of the granules, the low amylopectin crystallinity in the granule, and the low gel temperature and enthalpy observed in the differential scanning calorimeter. High final viscosity is achieved as soon as amylose is released from the pellet by heating in excess water and mixing. These RVA characteristics are unique to cereal starch and provide a new source of starch for food and industrial applications where low pasting viscosity and still high final viscosity are required.

Observations on gelatinization temperature in DSC are reflected in the results of studies of x-ray diffraction. Granules 292 and 342 have reduced crystallinity levels and crystalline shifts from type A typical of cereal starches to a mixture of types V and B. Type V is typical of amylose and reflects the amylose component of starch, which is complexed with fatty acids, while form B originates from amylopectin and predominantly reflects the content of residual starch amylopectin (Buleon et al., 1998).

An analysis of the genetic basis of mutations 292 and 342 demonstrates that these mutations are simple recessive mutations that give typical Mendelian relationships in outcrossing experiments. Crossbreeding studies have shown that 292 and 342 are allelic. Further analysis of the interaction between 292 and other shriveled endosperm mutations in the crossing experiments showed that the 292/342 mutations are also allelic with the Sex6 mutation in the MK6827 line. This mutation was mapped earlier, and it was shown that it is localized within 3 cM from the centromere of the short arm of chromosome 7H (Netsvetaev, 1990, Netsvetaev and Krestinkov, 1993, Biyashev et al., 1986, Netsvetaev, 1992).

A double haploid population was created between the husked Tantangara barley and the skinless mutant 292, and the shriveled endosperm mutation was mapped on the short arm of the 7HS chromosome within 16 cm from the nud gene at a location identical to that on the Sex6 mutation map.

Localization of the gene in the site adjacent to the centromere, on the short arm of chromosome 7HS, indicates that the causative mutation (sex6) is in a gene other than the mutation that causes the high amylose phenotype in AC38 (amo 1), which is mapped to chromosome 1H (Schondelmeier et al ., 1992). This localization on the map suggested that one of the candidates for the gene interrupted by the sex6 / 292 mutation is starch synthase II, which is known to be localized in wheat on the same chromosome site (Yamamori and Endo, 1996, Li et al ., 1999b). An analysis of the sequence of mutants 292 and 342 showed that in this gene there was a mutation of the G transition to A, which could cause a shortening of this gene, so that the C-terminal region containing the active site of the enzyme should not be translated, which mainly leads to the synthesis of a completely inactive squirrel. In addition, sequencing of the SSII gene from MK6827 showed a mutation of the G transition at A at position 242, which should also shorten this gene. This result confirms the allelic nature of mutations 292 and MK6827.

Identification of mutations in the SSII gene led to the development of a PCR marker diagnostic for a mutation in 292. This PCR marker was evaluated through 91 descendants of the 292 × Tantangara population, and it was shown that it is 100% cosegregated with a wrinkled endosperm phenotype and a reduced distribution chain length phenotype starch. The detection of allelic mutations in SSII genes from barley of various genetic backgrounds (292 and MK6827) that cause similar phenotypes, and the exact linkage of this mutation to the wrinkled grain phenotype gives a high degree of certainty that these mutations are present in the SSII 292, 342 and MK6827 genes are causative mutations leading to a sign of a shriveled endosperm.

The phenotype observed here for the SSII mutation in barley is in some respects similar to the phenotypes of SSII mutations in other plants, however, SSII mutations do not cause such a high content of amylose as the mutations found in 292/342. SSII mutations are known in peas (rug5, Craig et al., 1998) and Chlamydomonas (Fontaine et al., 1993) and lead to amylopectins with reduced chain length distributions, as observed here. There is also evidence to suggest that the Shrunken-2 mutation in maize is the result of a mutation of the SSII gene, although this still needs to be conclusively demonstrated (Harn et al., 1998, Knight et al., 1998). In maize, the Shrunken-2 mutation results in the formation of starches having lower gelatinization temperatures (Campbell et al., 1994). In wheat, Yamamori developed a triple nullisomic line that lacks the Sgp-1 protein (Yamamori, 1998), which, as shown by Li et al (Li et al., 1999b), is a product of the SSII gene. In wheat, the amylose content is increased to about 35%, and abnormal starch granules, altered crystallinity, and altered gelation temperature are observed (Yamamori, 1998). Differences in properties between barley SSII mutants and SSII mutants from other species are completely unexpected.

It was shown that the SSII mutation is capable of transfer by crossing from one genetic background to another and gives diagnostic morphology and grain composition typical of the initial 292, 342 and MK6827. In table (9), parameter data from 292 x Tantangara double haploid lines with respect to L / T, β-glucan content, distribution of chain lengths, RVA, and flour swelling volume demonstrate that lines carrying mutation 292 exhibit phenotypes typical of parent 292 In the following confirmation, segregation of seed from offspring from self-pollination of the second backcrossing 292 on Sloop showed a segregation ratio matching 3: 1 segregation for normal (74 seeds with L / T ratio <3.5) and shriveled phenotypes (21 seeds with L / T ratio> 3.5).

The accessibility of the SSII gene sequence and barley transformation systems provides the means necessary to turn off the SSII gene using gene suppression techniques in order to obtain a phenotype comparable to that found in SSII mutations. A recently developed highly effective strategy is to obtain a hairpin construct designed to produce double-stranded RNA, which should suppress the endogenous activity of SSII. Although complete shutdown mutations, similar to those described here, will be of interest, the use of DNA constructs with different promoters and the isolation of transgenes with different levels of expression of the hairpin construct should allow us to evaluate the effect of titration of the expression of this gene from normal levels to completely turned off levels.

It was shown that these mutations are capable of transferring from 292 to alternative genetic backgrounds of barley, while retaining the essential features of the original mutation 292. Tables 9 and 10 present phenotypic data for 292 × Tantangara double haploid progeny and seed from the second backcross with Sloop, and they indicate that these phenotypes are carried through the crossbreeding process.

Table 1 Barley Grain Composition Starch Content (%) ^a The content of amylose by HPLC (%) ^b Amylose content by binding of iodine (%) Protein Content (%) ^a β-glucan (%) ^a Total dietary fiber ^a (%) The insoluble dietary fiber ^as (%) Soluble dietary fiber ^a (%) Glacier but. 31,0 but. 11.5 4.3 21.6 16.6 5 AC38 47 47.4 60.6 10,4 5.8 24.9 28.8 6.1 Himalaya 49 25 25,4 10.0 4.8 27.1 18.1 9 292 17.7 71 68.9 15.0 9.5 30.3 21,4 8.9 342 21.9 62.5 71.7 15.7 8.3 28.3 19,4 8.9 MK6827 10,2 but. 44,4 21.3 but. but. but. but. Waxiro 42.8 but. 5,0 14.6 but. 19.8 12.7 7.1 Tantangara 51.6 but. 29.5 14.6 but. 17,2 12.7 4,5 ^a % of the mass of grain, 14% humidity
^b% of total starch content
but. not determined table 2 Grain sizes Grain mass (mg) Grain length (mm) Grain width (mm) Grain thickness (mm) L / T ratio Himalaya 51.01 ± 6.63 ^a 7.01 ± 0.51 3.58 ± 0.34 2.82 ± 0.36 2.48 Tantangara 50.40 ± 6.51 ^a 7.22 ± 0.98 3.60 ± 0.25 2.73 ± 0.21 2.64 Waxiro 45.71 ± 5.21 7.54 ± 0.47 3.40 ± 0.20 2.67 ± 0.19 2.82 AC38 50.79 ± 8.22 7.62 ± 0.65 3.35 ± 0.27 2.64 ± 0.25 2.89 292 32.13 ± 4.67 ^a 7.05 ± 0.49 3.63 ± 0.55 1.58 ± 0.20 4.46 342 35.45 ± 6.01 7.28 ± 0.55 3.76 ± 0.38 1.75 ± 0.18 4.16 MK6827 44.89 ± 3.78 11.20 ± 0.58 3.63 ± 0.27 1.77 ± 0.33 6.33 N = 50, except as indicated by ^a , where n = 200

Table 3 The distribution of chain lengths of starches in which the branching is removed by isoamylase D ^a Himalaya% ^b Tantangara% ^b AC38% ^b 342% ^b 292% ^b MK6827% ^b DP 6-11 24.15 22.40 26.33 38.18 38.96 37.98 DP 12-30 69.12 67.59 67.62 54.14 53.42 55.60 DP 31-60 6.73 10.01 6.05 7.68 7.62 6.42 ^{and the} degree of polymerization
^b % distribution of molar-based oligosaccharides Table 4 Thermal parameters of barley starch measured using DSC Peak 1 Peak 2 Start Peak the end ΔН Start Peak the end ΔН Glacier 55,4 59.3 65.3 4.2 93.9 101,4 107.7 0.87 AC38 55.0 62,2 68,2 3.9 89.3 100.1 106.9 1,195 Himalaya 56.8 60.9 68.0 4,5 93.1 101.8 108.3 0.78 292 46.0 51,2 58.1 0.29 88.7 97.7 104.9 1.34 342 45,2 50,4 56.8 0.47 86.5 97.0 105.0 1,59 Table 5 RVA Parameters for Barley Starch Peak viscosity Destruction Retention force Delay Final viscosity Normalized final viscosity * Pasting temperature (° C) Himalaya 871.5 653.1 218.4 235.8 454.2 926 64.9 Namoi 621.7 367.5 254.2 375.3 629.5 1284 65.9 AC38 226.7 87.3 139.4 188.4 327.8 697 68.9 292 92.1 ^** *** 133.9 230 363.9 2055 89.5 342 110.9 ^** *** 144.9 264.5 409.4 1869 87.9 MK6827 18.2 ^** *** 25.7 43.3 69 676 but. * Final viscosity divided by starch content of whole grain flour
** The value recorded at the time of peak viscosity for Himalaya
*** The value was below 0
but. not determined Table 6 Starch crystallinity data Sample % H ₂ O (W. B.) Crystallinity% ^* % ^* In% ^* V% ^* 292 29.6 9 - 13 87 342 35.8 12 - eighteen 81 AC38 26.1 19 93 7 (traces) Himalaya 27.7 27 93 7 (traces) Waxiro 29.7 41 94 6 - ^* (± 5%) Table 7 Offspring analysis Crossbreeding Shriveled Stuffed The calculated χ ² value ⁱⁿ 292 × Sloop ^a 45 155 χ ² (3: 1) = 1.0 292 × Tantangara ^B 45 46 χ ² (1: 1) = 0.01 ^and offspring from standard crosses
^b double haploid offspring
ⁱⁿ each case, χ ² (0.05), df = 1 = 3.84, therefore, the 292 × Sloop population corresponds to 3: 1 segregation, and the double 292 × Tantangara haploid population corresponds to 1: 1 segregation

Table 8 Assessment of 292xTantangara Double Haploid Lines Line number ^a Husk ^b Seed mass (mg) L / T ratio ⁱⁿ DP 6-11 (%) ^g Amylose content ^d PCR ^e one N 26 3.8 35.87 50,2 292 2 N 24 4.21 36.87 56.2 292 3 N 43 3.32 25.45 18.3 Wt 5 N 40 4,58 39.47 55.5 292 7 N 34 4.28 19.63 43.0 292 8 N 48 3.02 21.6 46.7 Wt 9 N 31 2.76 22.89 25.9 Wt 10 N 26 3.02 27.56 21.1 Wt eleven N 34 3,55 37.90 44.7 292 12 N fifty 2.94 26.37 32.8 Wt 13 N 27 4.29 38.68 48,4 292 fourteen N 56 3.07 22.98 20.8 Wt fifteen N 46 2.74 24.88 22.9 Wt 16 N 43 2.78 25.40 18.3 Wt 17 N 31 3.8 37.37 54,2 292 eighteen N 31 4,51 37.46 57.5 292 19 N 26 3,1 29.57 22.7 Wt twenty N 53 3.04 25.42 23.8 Wt 21 N 31 4,5 38.51 59.1 292 22 N 27 4.63 37.25 27,2 292 23 N 47 2.73 24.11 21,2 Wt 24 N 27 4,58 36.89 42.0 292 26 N 35 3.57 19.50 15.1 Wt 27 N 22 4.3 36.81 48.6 292 28 N 31 4.34 38.88 37.0 292 thirty N thirty 4.04 38.05 48,4 292 31 N 23 4.25 37.07 51.7 292 32 N 48 2.62 20.67 13.0 Wt 33 N 25 4.92 35.68 33.3 292 34 N 31 4.01 38.34 46.1 292 35 N 43 3.16 20.07 23.6 Wt 36 N 26 4.33 36.93 29.7 292 38 N 38 3.01 21.11 9.1 Wt 39 N 33 2.92 20.49 23.5 Wt 40 N 36 2.99 19.57 2.2 Wt 41 N thirty 4.05 37.82 40.9 292 42 N 47 2.95 20.80 11.9 Wt 43 N 40 3.24 21.97 18.1 Wt 45 N 52 2.78 19.97 14.5 Wt 46 N 29th 4.44 35.87 32.1 292 47 N 35 3.69 36.34 92.9 292 48 N 31 2.54 20.27 13,4 Wt 49 N 54 2.94 22.29 19.3 Wt fifty N fifty 2.94 21.92 20.6 Wt 51 N 43 3.73 20.59 18.1 Wt 53 N 31 4.12 36.52 55.3 292 54 N 34 4.02 35.17 57.1 292 55 N 32 4.19 41.35 60,4 292 56 N 29th 3.17 21.48 18.1 Wt 57 N thirty 4.85 36.66 46.3 292 58 N 32 2.97 23.83 13.8 Wt 59 N 46 2.91 24.15 9.2 Wt 60 N 44 2.74 22.39 13.5 Wt 61 N 31 4.47 35.67 61.3 292 63 N 32 4.3 36.94 39,4 292 64 N 39 2.93 21.95 20.5 Wt 65 N 26 3.87 37.51 20.7 292 66 N thirty 4.03 36.89 48.7 292 67 N 36 3.17 20.24 14,4 Wt 68 N 43 2.65 22.53 8.4 Wt 69 N 32 3.93 36.34 54.7 292 70 N 43 2.77 22.28 17.6 Wt 71 N 29th 3.73 38.73 31.5 292 72 N 47 2.65 22.00 20.8 Wt 73 N 36 4.09 39.58 49.0 292 74 N 24 4.18 36.15 47.8 292 75 N 34 2.99 24.42 14.2 Wt 76 N 31 4.35 35.95 49.9 292 77 N 49 3.19 21.22 17.0 Wt 78 N 33 2.78 21.27 15.6 Wt 79 N 31 2.85 23.04 21,2 Wt 80 N 38 3.18 19.88 18.9 Wt 81 N 37 2.84 24.22 16,2 Wt 82 N 33 4.64 39,99 45.3 292 84 N 28 3.62 36.98 28.9 292 85 N 26 6.44 44,43 41.3 292 86 N 32 2.87 30.73 16.1 Wt 88 N 26 4.62 46.22 39.3 292 89 N 38 2.88 31.25 16.3 Wt 90 N 32 3.19 31.11 13.8 Wt 91 N 31 4.17 42.86 37.3 292 92 N 27 3.99 45.30 44.6 292 93 N 37 2.99 30.77 12.5 Wt 94 N 43 3.67 29.46 21.9 Wt 96 N 33 5.69 47.34 52,2 292 97 N 23 3.41 31.36 17.1 Wt 98 N 32 5.95 45.27 52,4 292 99 N 19 3.68 38.36 1.7 292 one hundred N 36 3,1 31.92 15.4 Wt 101 N 58 3.29 24.71 2.9 Wt ^a double haploid line 292 × Tantangara
^b husk phenotype: N - skinless; N - with husk
^a ratio L / T: the ratio of length to thickness ^{g the} percentage of chains in starch with remote branching of the chain in the range DP6-DP11, calculated on a molar basis as the percentage of chains eluted between DP6 and DP65
^d amylose content is determined by the value of the blue stain of iodine
^e evaluation of PCR: 292 - the PCR reaction gives a band that gives a band of 169 p. plus 103 bp after enzymatic hydrolysis of NIaIV; Wt - the PCR reaction gives a band that gives bands 111 p.p., 103 p.p. and 57 bp after enzymatic hydrolysis of NlaIV.

Table 9 Detailed analysis of double haploid lines Line L / T ratio Face peak viscosity RVA (RVA units) final viscosity RVA (units RVA) peak / final viscosity ratio β-glucan content (%) flour swelling volume The control Sloop 2.78 23.5 535.8 483.5 1,11 2,3 7.54 Tantangara 2.64 22.4 507 395.1 1.28 5.16 5.97 Himalaya 2.48 24.2 873.9 449.3 1.94 8.53 8.18 AC38 2.89 26.33 226.7 327.8 0.69 5.8 3.75 292 4.46 38.9 92.1 363.9 0.25 13.09 2.00 MK6827 6.33 37.98 18.2 69 0.26 but. 2.11 Double haploid line Wild type 8 3.02 21.6 527.9 431.3 1.22 8.9 6.47 43 3.24 25,4 566.6 527.4 1,07 7.77 6.04 56 3.17 24.9 703.1 523.5 1.34 7.81 6.95 58 2.97 27.9 726.8 588.8 1.23 9.65 6.23 59 2.91 27.0 655 435.8 1,50 7.16 7.21 68 2.65 22.5 876.3 465.5 1.88 8.87 8.63 101 3.29 34.71 471.3 410.3 1.15 6.54 6.26 Mutant SSII 5 4,58 39.5 68.7 316.6 0.217 9.87 2,55 eleven 3,55 48,2 51.5 240.8 0.21 8.36 2,58 13 4.29 38.7 43.7 265.5 0.16 11.13 2.92 27 4.30 36.8 20.3 96.6 0.21 13.11 2.71 thirty 4.04 38.05 57.3 251.1 0.23 10.56 2.27 31 4.25 37.1 17.6 124.5 0.14 11.35 2.48 33 4.92 35.7 11.7 83.5 0.14 7.22 2.13 36 4.33 36.9 14.5 93.6 0.15 7.20 2.20 46 4.44 35.9 31.3 175.8 0.18 10.02 2,32 91 4.17 42.9 35.8 189.5 0.19 11.3 2.43 but. not determined

Table 10 Flour swelling data for BC2F2 seed Line Swelling volume C5 / 1 Plant 1 L: T> 3.5 2,118 C5 / 1 Plant 1 L: T <3.5 6,913 65/2 Plant 1 L: T> 3.5 2,382 65/2 Plant 1 L: T <3.5 7,565 65/2 Plant 2 L: T> 3.5 2,409 65/2 Plant 2 L: T <3.5 6,707

EXAMPLE 2

Vector design and construction

Plots of the barley SSII gene (as defined in FIG. 15) were cloned into transformation vectors. Three constructs were obtained for each target gene for the purpose of gene suppression strategy, (1) semantic co-suppression, (3) antisense, and (3) duplex-mediated suppression.

Figure 16 illustrates the sequence configuration in DNA constructs designed to suppress the expression of an endogenous target gene. The promoter can be selected either from endosperm-specific (such as the high molecular weight glutenin promoter, High Molecular Weight Glutenin, the SSI wheat promoter, the BEII wheat promoter), or from non-endosperm specific promoters (such as ubiquitin or 35S). The construct may also contain other elements that enhance transcription, such as the nos 3 OCS element. Illustrated DNA regions should be included in vectors containing suitable sequences of selectable marker genes and other elements, or in vectors that are co-transformed with vectors containing these sequences.

Cereal transformation

Methods of barley transformation (Tingay et al., 1997; Wan et al., 1994), oats (Somers et al., 1992, 1994; Gless et al., 1998; Zhang et al., 1999, Cho et al., 1999 ) and rye (Castillo et al., 1994; Pena et al., 1984) are described and can be used to transfer DNA constructs to produce transgenic plants.

Transgenic Plant Analysis

Identification of transgenic plants is carried out by identifying DNA construct DNA using PCR or Southern hybridization. The expression levels of individual barley starch biosynthesis genes are measured both at the mRNA level and at the protein level using standard techniques such as Northern hybridization and Western blotting, respectively. The content and composition of grain and starch are measured using standard methods, such as those described in Example 1.

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Claims (33)

1. A grain obtained from a barley plant containing the introduced mutation in the SSII gene or transgene, which lower the level of activity of SSII, and this grain is useful for industrial production of food products. 2. The grain according to claim 1, containing at least 6% β-glucan, expressed as a percentage of the total mass of unshelled grain. 3. The grain according to claim 1, which is a crushed, crushed grain, in the form of pearl barley, in the form of cereal flakes, crushed, chopped or whole grain. 4. The grain according to claim 1, having a characteristic selected from the group consisting of the presence of a starch content of more than 12% of seedless grain, the ability to grind lightly, the absence of purple color, bloodlessness and the presence of a length to thickness ratio of less than about 5.8. 5. The grain according to claim 1, having a length to thickness ratio of less than about 5.5. 6. The grain according to claim 1, having a length to thickness ratio in the range of 4 to 5. 7. The grain according to claim 1, which contains a transgene and the transgene is selected from the group consisting of DNA that directs the synthesis of an antisense molecule that interferes with the transcription or processing of the SSII gene, SSII-specific ribozyme, extra copies of the SSII gene for cosuppression of the SSII gene and DNA that directs the synthesis of a double-stranded RNA product that mediates suppression of the SSII gene. 8. A grain obtained from a barley plant, containing the introduced mutation in the SSII gene or transgene, which lower the level of activity of SSII, and which is useful for the industrial production of food products, and starch from this grain contains at least 50% (wt./wt. ) amyloses as measured by HPLC. 9. Starch obtained from grain according to any one of claims 1 to 8. 10. The starch according to claim 9, which exhibits a reduced gelatinization temperature, as measured by differential scanning calorimetry. 11. The starch of claim 10, the low gelatinization temperature of which is characterized by a lower temperature of the beginning of the first peak, determined using differential scanning calorimetry. 12. The starch of claim 10, the low gelatinization temperature of which is characterized by a lower peak temperature of the first peak, determined using differential scanning calorimetry. 13. The starch of claim 10, the low gelatinization temperature of which is characterized by a reduced enthalpy (ΔH) of the first peak determined using differential scanning calorimetry. 14. The starch according to claim 9, having a swelling volume of less than 3.2. 15. The starch of claim 14, having a swelling volume of at least about 2. 16. The starch according to claim 9, which when gelatinization exhibits a reduced peak viscosity. 17. The starch according to claim 9, the pasting temperature of which is higher than 75 ° C. 18. The starch according to claim 9, having a modified structure, and this changed structure is indicated by one or more than one characteristic selected from the group consisting of physical inaccessibility due to the presence of a high content of β-glucan, altered morphology of the granule, the presence of a noticeable starch-related lipid, low crystallinity and reduced chain length of amylopectin. 19. The starch according to claim 9, which contains at least 50% (wt./wt.) Amylose. 20. The starch according to claim 19, which contains at least 60% (wt./wt.) Amylose. 21. The starch according to claim 9, containing appreciable amounts of starch-bound lipid, the starch with bound lipid exhibiting a V-complex crystalline form, where this V-complex crystalline form is present in an amount of at least 10% of crystalline starch. 22. Starch according to item 21, which contains less than 5% of the A-complex form of starch. 23. The starch of claim 18, wherein the degree of crystallinity is reduced compared to normal starch isolated from barley, wherein the proportion of starch that exhibits crystallinity is less than about 20%. 24. The starch according to p. 18, which is characterized by a modified chain length of amylopectin after removal of the branching chain, and this changed chain length of amylopectin is characterized by one or more than one of the following: a) the proportion of chains having a length in the range from 6 to 11 residues is at least 25%, b) the proportion of chains having a length in the range from 12 to 30 residues is less than 65%, and c) the proportion of chains having a length in the range from 31 to 60 residues is less than 10%. 25. The starch according to paragraph 24, in which the proportion of amylopectin chains that have a length in the range of 31 to 60 residues is more than about 5%. 26. A barley plant having a reduced level of SSII activity, capable of producing grain according to any one of claims 1 to 8. 27. A method of obtaining a barley plant with a reduced level of activity of SSII, capable of producing grain according to any one of claims 1 to 8, including the stages at which a) introducing a genetic variation into the parent barley plant, which is either a mutation in the SSII gene or a transgene that lower the level of SSII activity, b) receive seed from this parent barley plant and C) carry out the selection of seeds obtained as a result of stage (b), for those seeds that have a reduced level of activity of SSII, and d) a barley plant is grown from seeds from stage (c). 28. The method according to item 27, in which stage (a) includes mutagenesis of the parent barley plant. 29. The method according to item 27, in which stage (C) includes selecting seeds for the phenotype of the shriveled endosperm or selecting seeds for high levels of amylose. 30. A method for producing starch, comprising the steps of: a) crushed grain according to any one of claims 1 to 8 and b) extract starch from this crushed grain. 31. Starch granules of barley obtained from grain according to any one of claims 1 to 8, and the starch of these granules contains at least 50% (wt./wt.) Amylose, as measured by HPLC. 32. Flour or wholemeal flour obtained from grain according to any one of claims 1 to 8, characterized in that it has a swelling volume of less than 3.2. 33. Flour or wholemeal obtained from grain according to any one of claims 1 to 8, characterized in that it has a swelling volume of at least about 2.

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