WO2005055704A2

WO2005055704A2 - Wheat having reduced waxy protein due to non-transgenic alterations of a waxy gene

Info

Publication number: WO2005055704A2
Application number: PCT/US2004/040779
Authority: WO
Inventors: Susan I. Fuerstenberg; Ann J. Slade
Original assignee: Arcadia Biosciences, Inc.
Priority date: 2003-12-03
Filing date: 2004-12-03
Publication date: 2005-06-23
Also published as: EP1708559A2; EP1708559A4; AU2004296834B2; US8735649B2; WO2005055704A3; EP1708559B1; CA2548030A1; US20050150009A1; AU2004296834A1; CA2548030C

Abstract

A series of independent non-transgenic mutations found at the waxy loci of wheat; wheat plants having these mutations in their waxy loci; and a method of creating and finding similar and/or additional mutations of the waxy by screening pooled and/or individual wheat plants. The wheat plants of the present invention exhibiting altered waxy activity in the wheat without having the inclusion of foreign nucleic acids in their genomes. The invention also includes food and non-food products as well as non-food products that incorporate seeds from the wheat plants having non-transgenic mutations in one or more waxy genes.

Description

WHEAT HAVING REDUCED WAXY PROTEIN DUE TO NON-TRANSGENIC ALTERATIONS OF A WAXY GENE

FIELD OF THE INVENTION This invention concerns non-transgenic mutations at one or more waxy locus of wheat and wheat plants having these non-transgenic mutations in their waxy sequences. This invention further concerns wheat plants having starch with lower amylose and higher amylopectin levels compared to starch from wild type wheat as a result of non- transgenic mutations in at least one of their waxy genes. This invention also concerns a method that utilizes non-transgenic means to create wheat plants having mutations in their waxy genes.

BACKGROUND The ratio of amylose to amylopectin in starch significantly affects the characteristics and quality of its finished food products including their digestibility_:, water retention, and resistance to staling. Starches that are high in amylose, a linear polymer, tend to gel when cooked whereas those that are high in amylopectin, a branching polymer, tend to form viscous pastes. Because of their unique physical properties including their pasting properties, solubility, gelling capacity, gel strength, swelling power, and vicosity, low amylose/high amylopectin starches are often used in the food industry to improve the texture and mouth-feel of select food products as well as their freeze-thaw stability. Amylose synthesis in a variety of plants, including wheat, is regulated for the most part by the enzyme granule-bound starch synthase (GBSSI), also known as waxy protein. The importance of this gene in starch synthesis has been well documented in naturally occurring varieties of GBSSI-deficient rice and corn, termed waxy mutants. Following the commercialization of waxy rice and waxy corn starches, there has been extensive interest by wheat breeders and the U.S. Department of Agriculture to develop . waxy wheat lines for use in the food industry as well as other commercial applications. Whereas starch from most traditional wheat cultivars is approximately 24% amylose and 76% amylopectin, starch from full waxy wheat lines (i.e., carrying deletions of all three genes) is almost 100% amylopectin. Potential commercial uses of waxy wheat starch include its use as a sauce thickener, emulsifier, and shelf-life extender. When mixed with traditional wheat flour in bread dough, waxy wheat flour improves crumb texture, freshness, and softness and eliminates the need for shortenings, thereby reducing fat content, unhealthy trans-fatty acids, and cost. Blended with other regular flours, waxy wheat flour improves the texture and tenderness of pasta and noodles, including Japanese udon noodles. In addition to the food industry, high amylopectin starches are important to the paper industry for enhancing the strength and printing properties of paper products and to the adhesive industry as a component of glues and adhesives, especially those used on bottles. Though breeding programs are underway to develop commercial varieties of waxy wheat, the polyploid nature of the wheat genome combined with homoeologous chromosome pairing has made the identification of waxy wheat mutants through traditional breeding methods difficult. The majority of wheat traded in commerce is Triticum aestivum or bread wheat. In this hexaploid, waxy is encoded by three homoeologues, Wx-7A, Wx-4A, and Wx-7D with the chromosomal locations 7 AS, 4AL, and 7DS (Murai et al., Isolation and characterization of the three Waxy genes encoding the granule-bound starch synthase in hexaploid wheat. Gene 234:71-79, 1999). In order to breed full waxy varieties using traditional breeding methods, knock-out mutations of all three homoeologues are required. Although several hundred lines of wheat have been identified that carry one or more mutations in the waxy genes Wx-4A and Wx-7A, only four deletion mutations of Wx-7D have been identified to date in over three thousand wheat lines that have been evaluated. One of these is in a Chinese landrace called Bai Huo, whose genetic heterogeneity makes it less suitable for traditional wheat breeding programs than modern elite cultivars. A cross between a double waxy null, Kantol07, and the Bai Huo landrace was performed to create the first full waxy null line in wheat (Nakamura et al., Mol Gen Genet 248:253-259, 1995). Despite the recent development of waxy breeding lines using this starting material, commercial varieties of waxy wheat are still not available, presumably due to the difficulty of removing undesirable agronomic traits from exotic germplasm. The paucity of Wx-7D deletion mutations has severely limited the development of commercial waxy wheat lines through traditional breeding. With the availability of the genetic sequences of the Triticum aestivum waxy genes, transgenic technology could be used to modify the expression of targeted proteins like waxy rather than rely on traditional breeding programs for the development of waxy wheat cultivars which could take years. However, public acceptance of genetically modified plants, particularly with respect to plants used for food, is low. Therefore, it would be useful to have additional commercial varieties of full or partial waxy wheat that were not the result of genetic engineering. The availability of multiple allelic mutations within each waxy locus would also allow for the breeding of new, diverse waxy phenotypes showing a spectrum of functional characteristics.

SUMMARY OF THE INVENTION In one aspect, this invention includes a wheat plant having reduced waxy enzyme activity compared to wild type wheat plants created by the steps of obtaining plant material from a parent wheat plant, inducing at least one mutation in at least one copy of a waxy gene of the plant material by treating the plant material with a mutagen to create mutagenized plant material, culturing the mutagenized plant material to produce progeny wheat plants, analyzing progeny wheat plants to detect at least one mutation in at least one copy of a waxy gene, selecting progeny wheat plants that have reduced waxy enzyme activity compared to the parent wheat plant; and repeating the cycle of culturing the progeny wheat plants to produce additional progeny plants having reduced waxy enzyme activity. In another aspect, this invention includes a wheat plant, flowers, seeds, plant parts, and progeny thereof having reduced waxy enzyme activity compared to the wild . type wheat plants wherein the reduced waxy enzyme activity is caused by a non- transgenic mutation in a waxy gene of the wheat plant. In another aspect, this invention includes a plant containing a mutated waxy gene, as well as flowers, seeds, pollen, plant parts and progeny of that plant. In another aspect, this invention includes food and food products as well as non-food products that incoφorate starch from wheat plants having reduced waxy enzyme activity caused by a non-transgenic mutation in the waxy gene. BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO: 1 shows the Triticum aestivum gene for starch synthase (waxy), complete cds. (GenBank Accession Number ABO 19623). This sequence corresponds to the waxy gene Wx-4A. SEQ ID NO: 2 shows the protein encoded by SEQ ID NO: 1 (GenBank Accession

Number BAA77351).

SEQ ID NO: 3 shows the Triticum aestivum gene for starch synthase (waxy), complete cds. (GenBank Accession Number ABO 19622). This sequence corresponds to the waxy gene Wx-7A. SEQ ID NO: 4 shows the protein encoded by SEQ ID NO: 3 (GenBank Accession

Number BAA77350).

SEQ ID NO: 5 shows the Triticum aestivum gene for starch synthase (waxy), complete cds. (GenBank Accession Number ABO 19624). This sequence corresponds to the waxy gene Wx-7D. SEQ ID NO: 6 shows the protein encoded by SEQ ID NO: 5 (GenBank Accession

Number BAA77352).

SEQ ID NO: 7-20 show the DNA sequences for the starch synthase (waxy) specific primers of the present invention. DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS The present invention describes a series of independent non-transgenic mutations in a waxy gene; wheat plants having these mutations in a waxy gene thereof; and a method of creating and identifying similar and or additional mutations in a waxy gene of wheat. Additionally, the present invention describes wheat plants created by this method having low amylose/high amylopectin starch without the inclusion of foreign nucleic acids in the plants' genomes. In order to create and identify the waxy mutations and the wheat plants of the present invention, a method known as TILLING was utilized. See McCallum et al, Nature Biotechnology, 18: 455-457, 2000; McCallum et al, Plant Physiology, 123: 439- 442, 2000; Colbert et al, Plant Physiol. 126(2):480-484, 2001 and US Patent No. 5,994,075 and 20040053236, all of which are incoφorated herein by reference. In the basic TILLING^® methodology, plant material, such as seeds, are subjected to chemical mutagenesis, which creates a series of mutations within the genomes of the seeds' cells. The mutagenized seeds are grown into adult Ml plants and self-pollinated. DNA samples from the resulting M2 plants are pooled and are then screened for mutations in a gene of interest. Once a mutation is identified in a gene of interest, the seeds of the M2 plant carrying that mutation are grown into adult M3 plants and screened for the phenotypic characteristics associated with the gene of interest. For the present invention the hexaploid cultivar Express (a hexaploid variety that naturally lacks the 4A locus) and the tetraploid cultivar Kronos were used. However, any cultivar of wheat having at least one waxy gene with substantial homology to SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5 may be used. The homology between the waxy genes and SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5 may be as low as 60% provided that the homology in the conserved regions of the gene is higher. One of skill in the art may prefer a wheat cultivar having commercial popularity or one having specific desired characteristics in which to create the waxy-mutated wheat plants. Alternatively, one of skill in the art may prefer a wheat cultivar having few polymoφhisms, such as an in-bred cultivar, in order to facilitate screening for mutations within the waxy locus. In one embodiment of the present invention, seeds from wheat plants were mutagenized and then grown into Ml plants. The Ml plants were then allowed to self- pollinate and seeds from the Ml plant were grown into M2 plants, which were then screened for mutations in their waxy loci. An advantage of screening the M2 plants is that all somatic mutations correspond to the germline mutations. One of skill in the art would understand that a variety of wheat plant materials, including but not limited to seeds, pollen, plant tissue or plant cells, may be mutagenized in order to create the waxy- mutated wheat plants of the present invention. However, the type of plant material mutagenized may affect when the plant DNA is screened for mutations. For example, when pollen is subjected to mutagenesis prior to pollination of a non-mutagenized plant, the seeds resulting from that pollination are grown into Ml plants. Every cell of the Ml plants will contain mutations created in the pollen, thus these Ml plants may then be screened for waxy mutations instead of waiting until the M2 generation. Mutagens that create primarily point mutations and short deletions, insertions, transversions, and or transitions (about 1 to about 5 nucleotides), such as chemical mutagens or radiation, may be used to create the mutations of the present invention. Mutagens conforming with the method of the present invention include, but are not limited to, ethyl methanesulfonate (EMS), methylmethane sulfonate (MMS), N-ethyl-N- nitrosurea (ENU), triethylmelamine (TEM), N-methyl-N-nitrosourea (MNU), procarbazine, chlorambucil, cyclophosphamide, diethyl sulfate, acrylamide monomer, melphalan, nitrogen mustard, vincristine, dimethylnitosamine, N-methyl-N'-nitro- Nitrosoguanidine (MNNG), nitrosoguanidine, 2-aminopurine, 7, 12 dimethyl- benz(a)anthracene (DMBA), ethylene oxide, hexamethylphosphoramide, bisulfan, diepoxyalkanes (diepoxyoctane (DEO), diepoxybutane (BEB), and the like), 2-methoxy- 6-chloro-9[3-(ethyl-2-chloro-ethyl)aminopropylamino] acridine dihydrochloride (ICR- 170), and formaldehyde. Spontaneous mutations in a waxy gene that may not have been directly caused by the mutagen can also be identified using the present invention. Any method of plant DNA preparation known to those of skill in the art may be used to prepare the wheat plant DNA for waxy mutation screening. For example, see Chen & Ronald, Plant Molecular Biology Reporter 17: 53-57 (1999); Stewart & Via, Bio Techniques, 1993, 14: 748-749. Additionally, several commercial kits are available, including kits from Qiagen (Valencia, CA) and Qbiogene (Carlsbad, CA). Prepared DNA from individual wheat plants was then pooled in order to expedite screening for mutations in a waxy gene of the entire population of plants originating from the mutagenized plant tissue. The size of the pooled group is dependent upon the sensitivity of the screening method used. Preferably, groups of two or more individuals are pooled. After the DNA samples were pooled, the pools were subjected to waxy sequence- specific amplification techniques, such as Polymerase Chain Reaction (PCR). For a general overview of PCR, see PCR Protocols: A Guide to Methods and Applications (Inns, M., Gelfand, D., Sninsky, J., and White, T., eds.), Academic Press, San Diego (1990). Any primers specific to the waxy loci or the sequences immediately adjacent to the waxy loci may be utilized to amplify the waxy sequences within the pooled DNA sample. Preferably, the primer is designed to amplify the regions of the waxy loci where useful mutations are most likely to arise. Most preferably, the primer is designed to detect exonic regions of the waxy genes. Additionally, it is preferable for the primer to avoid known polymoφhic sites in order to ease screening for point mutations. To facilitate detection of PCR products on a gel, the PCR primer may be labeled using any conventional labeling method. In the present invention, primers were designed based upon the waxy sequences, GenBank accession numbers AB019623 (SEQ ID NO: 1), AB019622 (SEQ ID NO: 3), and AB019624 (SEQ ID NO: 5). Exemplary primers (SEQ ID NOs: 7-20) that have proven useful in identifying useful mutations within the waxy sequences are shown below in Table 1.

The PCR amplification products may be screened for waxy mutations using any method that identifies nucleotide differences between wild type and mutant sequences. These may include, for example but not limited to, sequencing, denaturing high pressure liquid chromatography (dHPLC), constant denaturant capillary electrophoresis (CDCE), temperature gradient capillary electrophoresis (TGCE) (Li et al, Electrophoresis, 23(10):1499-1511, 2002, or by fragmentation using enzymatic cleavage, such as used in the high throughput method described by Colbert et al, Plant Physiology, 126:480-484, 2001. Preferably the PCR amplification products are incubated with an endonuclease that preferentially cleaves mismatches in heteroduplexes between wild type and mutant sequences. Cleavage products are electrophoresed using an automated sequencing gel apparatus, and gel images are analyzed with the aid of a standard commercial image-processing program. Each mutation is evaluated in order to predict its impact on protein function (i.e., completely tolerated to loss-of-function) using biofinormatics tools such as SIFT (Sorting Intolerant from Tolerant; Ng and Henikoff, Nuc Acids Res 31 :3812-3814, 2003), PSSM (Position-Specific Scoring Matrix; Henikoff and Henikoff, Comput ApplBiosci 12:135-143, 1996) and PARSESNP (Taylor and Greene, Nuc Acids Res 31:3808-3811, 2003). For example, a SIFT score that is less than 0.05 and a large change in PSSM score (roughly 10 or above) indicate a mutation that is likely to have a deleterious effect on protein function. Mutations that reduce waxy protein function are desirable. Because of the diverse ways in which wheat starch is used, an allelic series of mutations that result in a spectrum of functional characteristics would be useful. Preferred mutations include missense, splice junction, and nonsense changes including mutations that prematurely truncate the translation of the waxy protein from messenger RNA, such as those mutations that create a stop codon within the coding region of the waxy gene. These mutations include insertions, repeat sequences, modified open reading frames (ORFs) and, most preferably, point mutations. Once an M2 plant having a mutated waxy sequence is identified, the mutations are analyzed to determine its affect on the expression, translation, and/or activity of the waxy enzyme. First, the PCR fragment containing the mutation is sequenced, using standard sequencing techniques, in order to determine the exact location of the mutation in relation to the overall waxy sequence. If the initial assessment of the mutation in the M2 plant appears to be of a useful nature and in a useful position within the waxy sequence, then further phenotypic analysis of the wheat plant containing that mutation is pursued. First, the M2 plant is backcrossed or outcrossed twice in order to eliminate background mutations. Then the backcrossed or outcrossed plant is self-pollinated in order to create a plant that is homozygous for the waxy mutation. Waxy mutant plants are assessed to determine if the mutation results in a useful phenotypic change including starch type and content, starch characteristics, and seed opaqueness (for example, see Fujita et al., Plant Science, 160:595-602, 2001). The following mutations in Tables 2 are exemplary of the mutations created and identified according to the present invention. They are offered by way of illustration, not limitation.

Table 2: Examples of mutations created and identified in the Wx-4A, Wx-7A and Wx- 7D waxy homoeologs of wheat. Table 2

EXAMPLE 1 Mutagenesis In one embodiment of the present invention, wheat seeds of the hexaploid cultivar (Triticum aestivum) Express and the tetrapolid cultivar (Triticum turgidum, Durum) Kronos were vacuum infiltrated in H O (approximately 1000 seeds/100 ml H₂O for approximately 4 minutes). The seeds were then placed on a shaker (45 rpm) in a fume hood at ambient temperature. The mutagen ethyl methanesulfonate (EMS) was added to the imbibing seeds to final concentrations ranging from about 0.75% to about 1.2% (v/v). Following an 18-hour incubation period, the EMS solution was replaced with fresh H₂O (4 times). The seeds were then rinsed under running water for about 4-8 hours. Finally, the mutagenized seeds were planted (96/tray) in potting soil and allowed to germinate indoors. Plants that were four to six weeks old were transferred to the field to grow to fully mature Ml plants. The mature Ml plants were allowed to self-pollinate and then seeds from the Ml plant were collected and planted to produce M2 plants. DNA Preparation DNA from these M2 plants was extracted and prepared in order to identify which M2 plants carried a mutation at their waxy loci. The M2 plant DNA was prepared using the methods and reagents contained in the Qiagen^® (Valencia, CA) DNeasy^® 96 Plant Kit. Approximately 50 mg of frozen plant sample was placed in a sample tube with a tungsten bead, frozen in liquid mtrogen and ground 2 times for 1

minute each at 20 Hz using the Retsch^® Mixer Mill MM 300. Next 400 μl of solution

API [Buffer API, solution DX and RNAse (100 mg/ml)] at 80° C was added to the sample. The tube was sealed and shaken for 15 seconds. Following the addition of

130 μl Buffer AP2, the tube was shaken for 15 seconds. The samples were placed in

a freezer at minus 20° C for at least 1 hour. The samples were then centrifuged for 20

minutes at 5600 X g. A 400 μl aliquot of supernatant was transferred to another

sample tube. Following the addition of 600 μl of Buffer AP3/E, this sample tube was

capped and shaken for 15 seconds. A filter plate was placed on a square well block and 1ml of the sample solution was applied to each well and the plate was sealed.

The plate and block were centrifuged for 4 minutes at 5600 X g. Next, 800 μl of

Buffer AW was added to each well of the filter plate, sealed and spun for 15 minutes at 5600 X g in the square well block. The filter plate was then placed on a new set of

sample tubes and 80 μl of Buffer AE was applied to the filter. It was capped and

incubated at room temperature for 1 minute and then spun for 2 minutes at 5600 X g. This step was repeated with an additional 80 μl Buffer AE. The filter plate was removed and the tubes containing the pooled filtrates were capped. The individual

samples were then normalized to a DNA concentration of 5 to 10 ng/μl.

TILLING^® The M2 DNA was pooled into groups of two individual plants. The DNA

concentration for each individual within the pool was approximately 0.8 ng/μl with a

final concentration of 1.6 ng/ μl for the entire pool. Then, 5 μl of the pooled DNA

samples 8 ng was arrayed on microtiter plates and subjected to gene-specific PCR. PCR amplification was performed in 15 μl volumes containing 2.5 ng pooled

DNA, 0.75X ExTaq buffer (Panvera^®, Madison, WI), 2.6 mM MgCl₂, 0.3mM dNTPs,

0.3 μM primers, and 0.05U Ex-Taq (Panvera^®) DNA polymerase. PCR amplification

was performed using an MJ Research^® thermal cycler as follows: heat denaturation at 95° C for 2 minutes; followed by 8 cycles of "touchdown PCR" (94° C for 20 second, an annealing step starting at 70-68° C for 30 seconds and decreasing 1° C per cycle, a temperature ramp increasing 0.5° C per second to 72° C, and 72° C for 1 minute); then 25-45 cycles of PCR (94° C for 20 seconds, 63-61° C for 30 seconds, ramp of 0.5° C per second up to 72° C, 72° C for 1 minute); and finally extension, denaturation and reannealing steps (72° C for 8 minutes; 98° C for 8 minutes; 80° C for 20 seconds, followed by 60 cycles of 80° C for 7 seconds decreasing 0.3 degrees/cycle). The PCR primers (MWG Biotech, Inc., High Point, NC) were mixed as follows:

2.5 μl 100 μM IRD-700 labeled left primer 7.5 μl 100 μM left primer

9.0 μl 100 μM JRD-800 labeled right primer 1.0 μl 100 μM right primer A label can be attached to each primer as described or to only one of the primers. Alternatively, Cy5.5 modified primers could be used. The JRD-label was coupled to the oligonucleotide using conventional phosphoramidite chemistry. PCR products (15 μl) were digested in 96-well plates. Next, 30 μl of a

solution containing 10 mM HEPES [4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid] (pH 7.5), 10 mM MgSO₄, 0.002% (w/v) Triton^® X-100, 20 ng/ml of bovine serum albumin, and CEL 1 (Transgenomic^®, Inc.; 1:100,000 dilution) was added with mixing on ice, and the plate was incubated at 45° C for 15 min. The specific activity

of the CELl was 800 units/μl, where a unit was defined by the manufacturer as the

amount of enzyme required to produce 1 ng of acid-soluble material from sheared, heat denatured calf thymus DNA at pH 8.5 in one minute at 37 ° C. Reactions were

stopped by addition of 10 μl of a 2.5 M NaCl solution with 0.5 mg/ml blue dextran

and 75 mM EDTA, followed by the addition of 80 μl isopropanol. The reactions

were precipitated at 80° C, spun at 4000 rpm for 30 minutes in an Eppendorf

Centrifuge 5810. Pellets were resuspended in 8 μl of 33% formamide with 0.017%

bromophenol blue dye, heated at 80 ° C for 7 minutes and then at 95 ° C for 2 minutes. Samples were transferred to a membrane comb using a comb-loading robot (MWG Biotech). The comb was inserted into a slab acrylamide gel (6.5%), electrophoresed for 10 min, and removed. Electrophoresis was continued for 4h at 1,500-V, 40- W, and 40-mA limits at 50°C. During electrophoresis, the gel was imaged using a LI-COR^® (Lincoln, NE) scanner which was set at a channel capable of detecting the JR Dye 700 and 800 labels. The gel image showed sequence-specific pattern of background bands common to all 96 lanes. Rare events, such as mutations, create new bands that stand out above the background pattern. Plants with bands indicative of mutations of interest were evaluated by TILLING^® individual members of a pool mixed with wild type DNA and then sequencing individual PCR products. Plants carrying mutations confirmed by sequencing were grown up as described above (e.g., the M2 plant was backcrossed or outcrossed twice in order to eliminate background mutations and self- pollinated in order to create a plant that was homozygous for the mutation). Mutations identified during TILLING^® are shown below in Tables 3 and 4. Table 3

Table 4

Phenotypic Analysis Phenotype was examined in M3 progeny of an Express line carrying mutations that were predicted by bioinformatics analysis to affect gene function. The mutations were a truncation mutation in Wx-7D (Q197*) which was predicted to result in premature termination of the protein and a missense mutation in the Wx-7A homoeolog (A468V) which was predicted to severely affect protein function by a SIFT score of 0.00 and a change in PSSM score of 16.4. Since the Express line lacks the Wx-4A homoeolog, progeny that were homozygous for both mutations were predicted to display nearly a full waxy phenotype. Iodine was used to stain the endosperm of the progeny. Waxy endosperm stains a reddish brown with iodine, whereas amylose-containing endosperm stains very dark blue (Nakamura et al, Mol. Gen. Genet. 248:253-259, 1995). Seeds were soaked in water for three hours, cut in half, and then treated with a four-fold dilution of iodine stain for 15 minutes. In contrast to seeds of the parental Express line that stained very dark blue, seeds of the double homozygous mutant stained very light blue with iodine indicating that amylose levels were significantly reduced by the mutations. These findings are consistent with the effect on protein function predicted by the mutations' SIFT and POSSUM scores. The above examples are provided to illustrate the invention but not limit its scope. Other variants of the invention will be readily apparent to one of ordinary skill in the art and are encompassed by the appended claims and all their equivalents. All publications, patents, and patent applications cited herein are hereby incorporated by reference.

Claims

We claim: 1. A method of creating a wheat plant exhibiting a reduced waxy enzyme activity compared to wild type wheat plants, comprising the steps of: a. obtaining plant material from a parent wheat plant; b. inducing at least one mutation in at least one copy of a deoxyhypusine synthase gene of the plant material by treating the plant material with a mutagen to create mutagenized plant material; c. culturing the mutagenized plant material to produce progeny wheat plants; d. analyzing progeny wheat plants to detect at least one mutation in at least one copy of a waxy gene; e. selecting progeny wheat plants that have reduced waxy enzyme activity compared to the parent wheat plant; and f. repeating the cycle of culturing the progeny wheat plants to produce additional progeny wheat plants having reduced waxy enzyme activity.

2. The method of claim 1 wherein the plant material is selected from the group consisting of seeds, pollen, plant cells, or plant tissue.

3. The method of claim 1 wherein the mutagen is ethyl methanesulfonate.

4. The method of claim 3 wherein the concentration of ethyl methanesulfonate used is from 0.75% to about 1.2%.

5. The method of claim 1 where the progeny wheat plant are analyzed by a. isolating genomic DNA from the progeny wheat plants; and b. amplifying segments of the waxy gene in the isolated genomic DNA using primers specific to the waxy gene or to the DNA sequences adjacent to the waxy gene.

6. The method of claims 1 and 5 wherein the waxy gene is substantially homologous SEQ. ID. No.: 1.

7. The method of claims 1 and 5 wherein the waxy gene is substantially homologous to SEQ.I.D. No.: 3.

8. The method of claims 1 and 5 wherein the waxy gene is substantially homologous to SEQ. I.D. No.: 5.

9. The method of claim 5 where at least one primer has a sequence substantially homologous to a sequence in the group consisting of SEQ. ID. NOs. 7 through 20.

10. The method of claim 1 wherein the mutation detected in step d is evaluated to determine the mutation's likelihood of having a deleterious effect on waxy enzyme activity.

11. The method of claim 10 where in the mutation is evaluated using a bioinformatics tool selected from the group consisting of SIFT, PSSM and PARSESNP.

12. A wheat plant created according to the method of claim 1.

13. The wheat plant of claim 12 having a low amylose/high amylopectin starch ratio compared to the parent wheat plant.

14. Flower, seeds, pollen, plant parts or progeny of the wheat plant of claim 12.

15. Parts of the seeds of claim 14.

16. Starch from a wheat plant created according to claim 12.

17. Food and food products incorporating any portion of the seed of the wheat plant of claim 12.

18. Paper products incorporating any portion of the seed of the wheat plant of claim 12.

19. Adhesive products incorporating any portion of the seed of the wheat plant of claim 12.

20. A wheat plant exhibiting a low amylose/high amylopectin starch ratio created by breeding a wheat plant with the wheat plant of claim 12.

21. An endogenous waxy gene having substantial homology to SEQ. I.D. No. 1 and having a non-transgenic mutation within the endogenous waxy gene following treatment with a mutagen.

22. The endogenous waxy gene of claim 21 wherein the non-transgenic mutation is a truncation mutation.

23. The endogenous waxy gene of claim 21 wherein the non-transgenic mutation is a splice junction mutation.

24. The endogenous waxy gene of claim 21 wherein the non-transgenic mutation is a missense mutation.

25. A wheat plant containing the endogenous waxy gene of claim 21.

26. Flowers, seeds, pollen, plant parts, and progeny of the waxy plant of claim 25.

27. Parts of the seeds of claim 26.

28. Starch from the wheat plant of claim 25.

29. Food and food products incorporating any portion of the seeds of the wheat plant of claim 25.

30. Paper products incorporating any portion of the seed of the wheat plant of claim 25.

31. Adhesive products incorporating any portion of the seed of the wheat plant of claim 25.

32. An endogenous waxy gene having substantial homology to SEQ. I.D. No. 3 and having a non-transgenic mutation within the endogenous waxy gene following treatment with a mutagen.

33. The endogenous waxy gene of claim 32 wherein the non-transgenic mutation is a truncation mutation.

34. The endogenous waxy gene of claim 32 wherein the non-transgenic mutation is a splice junction mutation.

35. The endogenous waxy gene of claim 32 wherein the non-transgenic mutation is a missense mutation.

36. A wheat plant containing the endogenous waxy gene of claim 32.

37. Flowers, seeds, pollen, plant parts, and progeny of the waxy plant of claim 36.

38. Parts of the seeds of claim 37.

39. Starch from the wheat plant of claim 36.

40. Food and food products incorporating any portion of the seeds of the wheat plant of claim 36.

41. Paper products incorporating any portion of the seed of the wheat plant of claim 36.

42. Adhesive products incorporating any portion of the seed of the wheat plant of claim 36.

43. An endogenous waxy gene having substantial homology to SEQ. I.D. No. 5 and having a non-transgenic mutation within the endogenous waxy gene following treatment with a mutagen.

44. The endogenous waxy gene of claim 43 wherein the non-transgenic mutation is a truncation mutation.

45. The endogenous waxy gene of claim 43 wherein the non-transgenic mutation is a splice junction mutation.

46. The endogenous wary gene of claim 43 wherein the non-transgenic mutation is a missense mutation.

47. A wheat plant containing the endogenous waxy gene of claim 43.

48. Flowers, seeds, pollen, plant parts, and progeny of the waxy plant of claim 47.

49. Parts of the seeds of claim 48.

50. Starch from the wheat plant of claim 47.

51. Food and food products incorporating any portion of the seeds of the wheat plant of claim 47.

52. Paper products incorporating any portion of the seed of the wheat plant of claim 47.

53. Adhesive products incorporating any portion of the seed of the wheat plant of claim 47.