Wheat cultivar ofree for improvement of gluten intolerance and wheat-dependent exercise-induced anaphylaxis

Abstract

The present invention relates to Ofree, a novel wheat cultivar produced by crossing Keumkang wheat and Olgeuru wheat, in which low-molecular-weight glutenin subunit (LMW-GS) alleles located at the Glu-B3 loci (major cause of gluten intolerance) and -5 gliadin genes (major cause of wheat-dependent exercise-induced anaphylaxis (WDEIA)) have been deleted; a method of developing a new wheat cultivar using Ofree; wheat flour produced from Ofree; a method of producing a processed food using the wheat flour; and a processed food produced by the aforementioned production method. It is expected that Ofree provided in the present invention can be widely used for the production of processed foods capable of preventing the onset of celiac disease caused by gluten intolerance and the onset of WDEIA.

Claims

1. A new wheat cultivar in which low-molecular-weight glutenin subunit (LMW-GS) alleles located at Glu-B3 loci have been deleted, the new wheat cultivar is deposited with a deposition number KACC88001BP.

2. A method of developing an improved wheat cultivar, the method including using the new wheat cultivar according to claim 1 as a father plant or a mother plant for crossing with another wheat cultivar.

3. The method of claim 2, wherein the other wheat cultivar is Jokyung wheat, Jopoom wheat, Baekjoong wheat, or Goso wheat.

4. Wheat flour obtained by milling the new wheat cultivar according to claim 1.

5. A food composition including the wheat flour according to claim 4.

6. The food composition according to claim 5, wherein the food composition is noodles, cookies, or bread.

7. A method of producing a processed food, the method including: (a) obtaining wheat flour by milling the new wheat cultivar according to claim 1; (b) preparing a dough containing the wheat flour; and (c) processing the dough.

8. The method according to claim 7, further comprising: mixing the wheat flour obtained from (a) with wheat flour obtained from another wheat cultivar to form mixed flour.

9. The method according to claim 8, wherein the wheat flour obtained from (a) is included in the mixed flour in an amount of 50 to 99% v/v.

10. The method according to claim 8, wherein the another wheat cultivar is selected from the group consisting of Keumkang wheat, Olgeuru wheat, Jokyung wheat, Jopoom wheat, Baekjoong wheat, Goso wheat, and combinations thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The above and other objects, features and advantages of the present invention will become more apparent to those of ordinary skill in the art by describing in detail exemplary embodiments thereof with reference to the accompanying drawings, in which:

(2) FIG. 1 is a genealogy diagram for illustrating the process of developing Ofree, a new wheat cultivar.

(3) FIG. 2A is an electrophoresis image for showing the results of polymerase chain reaction (PCR) amplification of genes located at the Glu-B3 loci in Keumkang, Olgeuru and Ofree.

(4) FIG. 2B is an electrophoresis image for showing HMW-GS- and LMW-GS-related proteins in Keumkang, Olgeuru and Ofree.

(5) FIG. 2C is an electrophoresis image for showing LMW-GS-related proteins in various amounts of an Ofree extract.

(6) FIG. 3 is an electrophoresis image for showing the results of detection of -5 gliadin proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of a gliadin fraction in Keumkang, Olgeuru and Ofree, wherein the first, second and third lanes represent Keumkang, Olgeuru and Ofree, respectively.

(7) FIG. 4 is an electrophoresis image for showing the results of detection of -5 gliadin proteins by acid polyacrylamide gel electrophoresis (A-PAGE) analysis of the glutenin fraction in Keumkang, Olgeuru, and Ofree, wherein the first, second and third lanes represent Ofree, Keumkang and Olgeuru, respectively.

(8) FIG. 5 shows the results of reversed-phase high-performance liquid chromatography (RP-HPLC) analysis of a gliadin fraction in Keumkang, Olgeuru and Ofree (top to bottom).

(9) FIG. 6 shows the immunoblotting results of -5 gliadin-specific antibodies in Keumkang, Olgeuru and Ofree.

(10) FIG. 7 shows the results of two-dimensional electrophoresis performed on all seed storage protein fractions in Keumkang, Olgeuru, Ofree (DH20) and Butte 86 followed by immunoblotting using the serum of a wheat-dependent exercise-induced anaphylaxis (WDEIA) patient; and the results of protein analysis by tandem mass spectrometry (MS/MS) superimposed on the immunoblotting results.

(11) FIG. 8 is a set of images for showing Ofree (DH20), Keumkang and Olgeuru on their heading date (A) and the grains (B) and bread pieces (C) of Ofree (DH20), Keumkang, and Olgeuru.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

(12) Hereinafter, the present invention will be described in greater detail with reference to examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1: Development of New Wheat Variety

(13) A novel wheat cultivar having high cold resistance and desirable growth habit was developed by artificially crossing Keumkang and Olgeuru to obtain a progeny wheat plant F1. Haploid of F1 was cultivated at CIMMYT in Mexico and testing the productivity of F1 for four years in Iksan. The novel wheat cultivar was named Ofree (FIG. 1).

(14) FIG. 1 is a genealogy diagram for illustrating the process of developing a new wheat cultivar Ofree.

Example 2: Genotype Identification of Ofree

Example 2-1. Confirmation of Deletion of Genes Located at Glu-B3 Loci

(15) The genetic characteristics of Ofree developed by the processes described in Example 1 were determined in terms of the expression levels of genes at the Glu-1 loci components of high-molecular-weight glutenin subunits (HMW-GSs) in gluten and genes at the Glu-3 loci that is components of low-molecular-weight glutenin subunits (LMW-GSs) in gluten.

(16) In brief, a genomic DNA extraction kit for plants (SolGent Co., Ltd., Korea) was used to extract genomic DNA from the young leaves of Keumkang, Olgeuru and Ofree. Subsequently, polymerase chain reaction (PCR) was carried out using the genomic DNA extracted earlier as a template and primers capable of detecting genes at the Glu-A1, Glu-B1, Glu-D1, Glu-A3, Glu-B3 or Glu-D3 loci (listed below) to determine which of the genes at the Glu-1 and Glu-3 loci were contained in the genetic composition of Ofree (Table 1).

(17) TABLE-US-00001 Glu-A1abF: (SEQIDNO:1) 5-AAGACAAGGGGAGCAAGGT-3 Glu-A1abR: (SEQIDNO:2) 5-GTGCTCCGCGCTAACATG-3 Glu-A1cF: (SEQIDNO:3) 5-ACGTTCCCCTACAGGTACTA-3 Glu-A1CR: (SEQIDNO:4) 5-TATCACTGGCTAGCCGACAA-3 Glu-B1bcfF: (SEQIDNO:5) 5-TTCTCTGCATCAGTCAGGA-3 Glu-B1bcfR: (SEQIDNO:6) 5-AGAGAAGCTGTGTAATGCC-3 Glu-D1dF: (SEQIDNO:7) 5-GCCTAGCAACCTTCACAATC-3 Glu-D1dR: (SEQIDNO:8) 5-GAAACCTGCTGCGGACAAG-3 Glu-D1adfF: (SEQIDNO:9) 5-TTTGGGGAATACCTGCACTACTAAAAAGGT-3 Glu-D1adfF: (SEQIDNO:10) 5-AAAAGGTATTACCCAAGTGTAACTTGTCCG-3 Glu-D1adfR: (SEQIDNO:11) 5-AATTGTCCTGGCTGCAGCTGCGA-3 Glu-A3aF: (SEQIDNO:12) 5-AAACAGAATTATTAAAGCCGG-3 Glu-A3bR: (SEQIDNO:13) 5-GGTTGTTGTTGTTGCAGCA-3 Glu-A3bF: (SEQIDNO:14) 5-55CAGATGCAGCCAAACAA-3 Glu-3AbR: (SEQIDNO:15) 5-GCTGTGCTTGGATGATACTCTA-3 Glu-A3acF: (SEQIDNO:16) 5-AACAGAATTATTAAAGCCGG-3 Glu-A3acR: (SEQIDNO:17) 5-CTGTGCTTGGATGATACTCTA-3 Glu-A3dF: (SEQIDNO:18) 5-TTCAGATGCAGCCAAACAA-3 Glu-A3dR: (SEQIDNO:19) 5-TGGGGTTGGGAGACACATA-3 Glu-A3eF: (SEQIDNO:20) 5-AAACAGAATTATTAAAGCCGG-3 Glu-A3eR: (SEQIDNO:21) 5-GGCACAGACGAGGAAGGTT-3 Glu-B3dF: (SEQIDNO:22) 5-CACCATGAAGACCTTCCTCA-3 Glu-B3dR: (SEQIDNO:23) 5-GTTGTTGCAGTAGAACTGGA-3 Glu-B3fgF: (SEQIDNO:24) 5-TATAGCTAGTGCAACCTACCAT-3 Glu-B3fgR: (SEQIDNO:25) 5-CAACTACTCTGCCACAACG-3 Glu-B3gF: (SEQIDNO:26) 5-CAAGAAATACTAGTTAACACTAGTC-3 Glu-B3gR: (SEQIDNO:27) 5-GTTGGGGTTGGGAAACA-3 Glu-B3hF: (SEQIDNO:28) 5-CCACCACAACAAACATTAA-3 Glu-B3hR: (SEQIDNO:29) 5-GTGGTGGTTCTATACAACGA-3 Glu-B3iF: (SEQIDNO:30) 5-TATAGCTAGTGCAACCTACCAT-3 Glu-B3iR: (SEQIDNO:31) 5-TGGTTGTTGCGGTATAATTT-3 Glu-D3abF: (SEQIDNO:32) 5-TTGGGCCTAATCGCTCGC-3 Glu-D3abR: (SEQIDNO:33) 5-TAGTCTCCATCTGCGCAATT-3 Glu-D3cF: (SEQIDNO:34) 5-CAGCTAAACCCATGCAAGC-3 Glu-D3cR: (SEQIDNO:35) 5-CAATGGAAGTCATCACCTCAA-3

(18) TABLE-US-00002 TABLE 1 Genetic composition of Glu-1 and Glu-3 Wheat cultivars HMW-GS LMW-GS and wheat lines Glu-A1 Glu-B1 Glu-D1 Glu-A3 Glu-B3 Glu-D3 Keumkang b b d c h a Olgeuru b b f d d a Ofree b b f c a

(19) As shown in Table 1, most of the genes expressed in one of Keumkang and Olgeuru were also expressed in Ofree, except for Glu-B3 (FIG. 2A).

(20) FIG. 2A is an electrophoresis image for showing the results of polymerase chain reaction (PCR) amplification of genes located at the Glu-B3 loci in Keumkang, Olgeuru and Ofree. As shown in FIG. 2A, genes located at the Glu-B3 loci were not detected from Ofree.

(21) Hence, for the purpose of detecting genes located at the Glu-B3 loci at the protein level, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of Keumkang, Olgeuru and Ofree extracts obtained from young leaves was carried out (FIG. 2B)

(22) FIG. 2B is an electrophoresis image for showing HMW-GS- and LMW-GS-related proteins in Keumkang, Olgeuru and Ofree. As shown in FIG. 2B, no gene was detected at the Glu-B3 loci of Ofree even at the protein level.

(23) In addition, to find out whether the results shown in FIG. 2B were obtained because of an insufficient amount of the Ofree extract used for SDS-PAGE, various amounts of the Ofree extract were subjected to SDS-PAGE (FIG. 2C).

(24) FIG. 2C is an electrophoresis image for showing LMW-GS-related proteins in various amounts of the Ofree extract. As shown in FIG. 2C, as the amount of the Ofree extract increased, the amount of various proteins detected also increased accordingly except for genes located at the Glu-B3 loci, which were still not detected even when a large amount of the Ofree extract was examined.

(25) To summarize the results shown in FIGS. 2A to 2C, since Ofree provided in the present invention is a wheat cultivar in which LMW-GS alleles located at the Glu-B3 loci (major cause of gluten intolerance) have been deleted, it can be used in the production of a bakery product that can be ingested by a person with gluten intolerance without causing side effects.

Example 2-2. Confirmation of Deletion of -5 Gliadin Genes in Ofree

(26) Gliadin extraction was performed by consulting a prior-art document (Dziuba M, Nacz D, Szerszunowicz I, Waga J. 2014. Proteomic analysis of wheat /A- and -gliadins. Czech J Food Sci 32: 437-442). 1 ml of a 0.15 M aqueous NaCl solution was mixed with 100 mg of each of finely pulverized Keumkang, Olgeuru, and Ofree. Each mixture was reacted for two hours at 25 C., centrifuged at the speed of 15,000 rpm at 20 C. for five minutes, and then a supernatant thereof was removed. The precipitate was mixed with 1 ml of a 70% aqueous ethanol solution, the mixture was reacted for 12 hours at a constant temperature and centrifuged at the speed of 15,000 rpm at 20 C. for five minutes, and then a supernatant of the mixture was stored at 20 C.

(27) Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) Analysis and Results

(28) A gliadin stored at 20 C. was mixed with a sample buffer (50 mM Tris-HCl, pH 6.8, 8% -mercaptoethanol, 2% SDS, 20% glycerol) in a ratio of 1:2 to produce a mixture, and 5 l of the mixture was loaded onto a 12.5% SDS-PAGE gel and subjected to electrophoresis at 70 V for 13 hours and at 110 V for four hours. After electrophoresis, the gel was stained with a Coomassie Brilliant Blue R-250 staining solution (Bio-Rad Laboratories, Inc.) for four hours and then decolorized with a decolorizing solution (10% glacial acetic acid, 10% methanol, 80% distilled water) for five hours, and the obtained protein band patterns were analyzed.

(29) According to the results shown in FIG. 3, no -5 gliadin was detected from Ofree, unlike the cases of Keumkang and Olgeuru.

(30) Acid Polyacrylamide Gel Electrophoresis (A-PAGE) Analysis and Results

(31) 1 ml of 70% ethanol was mixed with 100 mg of each of finely pulverized Keumkang, Olgeuru and Ofree. Each mixture was stirred at 25 C. for one hour, centrifuged at the speed of 14,000 rpm at a constant temperature for 10 minutes, and 500 l of a supernatant thereof was mixed with 250 l of a 5:1 solution of glycerin and 1% methyl violet.

(32) 10 l of a gliadin was added to a 12% gel solution (12% acrylamide, 0.6% bis-acrylamide, 0.1% ascorbic acid, 0.004% Iron (II) sulfate heptahydrate, 0.25% aluminum lactate, pH adjusted to 3.1 with lactic acid), and the mixture was loaded and subjected to electrophoresis at 400 V for five hours. Then, the gel was stained with a Coomassie Brilliant Blue R-250 staining solution for three hours and then decolorized with 10% acetic acid and 10% methanol for four hours, and the obtained protein band patterns were analyzed.

(33) According to the results shown in FIG. 4, no -5 gliadin was detected from Ofree-unlike the cases of Keumkang and Olgeuru.

(34) Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) Analysis and Results

(35) 1 ml of 70% ethanol was mixed with 100 mg of each of finely pulverized Keumkang, Olgeuru and Ofree. Each mixture was reacted at 25 C. for one day, centrifuged at the speed of 15,000 rpm at 20 C. for five minutes, and then a supernatant thereof was dissolved with 300 ml of 0.1% trifluoroacetic acid (TFA) in 20% acetonitrile (ACN) and filtered with a Whatman polyvinylidene fluoride (PVDF) syringe filter (0.45 m, Sigma-Aldrich Co. LLC). High-performance liquid chromatography (HPLC) was performed using an Agilent Zorbax 300SB-C18 column (300 pore size, 5 m particle size, 4.6 mm inner diameter, 150 mm length) and a Waters Alliance e2695 HPLC system. Solvents used for HPLC include solvent A (0.1% TFA in water), solvent B (0.1% TFA in ceric ammonium nitrate (CAN)), solvent C (MeOH) and solvent D (MeOH). For analysis, 10 l of each sample was injected and separations were accomplished with a linear gradient of 25 to 50% (solvent B) for 70 minutes and a flow rate of 1 ml/min, and a column temperature of 65 C. and a sample temperature of 15 C. were maintained. The analyzed samples were observed at 210 nm.

(36) FIG. 5 shows the results of RP-HPLC analysis of a gliadin fraction in Keumkang, Olgeuru and Ofree. As shown in FIG. 5, no -5 gliadin was detected from Ofree unlike the cases of Keumkang and Olgeuru.

(37) Results of Immunoblotting Using -5-Gliadin-Specific Antibodies

(38) Total protein extraction, two-dimensional electrophoresis and immunoblotting were performed by referencing a document (Susan B. Altenbach et al. 2015. Assessment of the allergenic potential of transgenic wheat (Triticum aestivum) with reduced levels of 5-gliadins, the major sensitizing allergen in wheat-dependent exercise-induced anaphylaxis. Journal of Agricultural and Food Chemistry. DOI: 10.1021). Total protein extraction from 100 mg of each of finely pulverized Keumkang, Olgeuru, and Ofree was performed using a 2% SDS sample buffer (2% SDS, 10% glycerol, 0.04 M Tris-HCl, pH 8.5, 50 mM DTT). Isoelectric focusing (IEF) was manually performed using an ampholyte (pI 3-10) and a capillary tube, and the second electrophoresis was performed on a NuPAGE 4-12% Bis-Tris protein gel. Proteins in two-dimensional gel electrophoresis (2-DE) spots were identified by tandem mass spectrometry (MS/MS). The MS/MS analysis was carried out by the method described in a document (Dupont, F M, Vensel W H, Tanaka C K, Hurkman II W J, Altenbach S B. 2011. Deciphering the complexities of the wheat flour proteome using quantitative two-dimensional electrophoresis, three proteases and tandem mass spectrometry. Proteome Science. 9(10). Available at: proteomesci.com). For each spot, three types of proteases (trypsin, chymotrypsin, thermolysin) were used to obtain maximum amino acid sequence coverage. The NCBI protein accession numbers obtained as a result of protein identification within each spot are provided in FIG. 6. -5 gliadin (BAE20328), i-type LMW-GS (AAS10189), s-type LMW-GS(BAD12055, AEI00677) and m-type LMW-GS(AEI00671) were identified from Keumkang, and -5 gliadins (BAE20328, AII26682), i-type LMW-GSs (AGM38903, ACY08811) and s-type LMW-GS (ACY08813) were identified from Olgeuru. After 2-DE, the total protein was transferred to a nitrocellulose membrane. The serum of the same patient was subjected to immunoblotting.

(39) As shown in FIG. 6, no -5 gliadin protein was produced in Ofree.

(40) Results of Immunoblotting Analysis Using Serum of WDEIA Patient

(41) Total protein extraction, two-dimensional electrophoresis and immunoblotting were performed by referencing a document (Susan B. Altenbach et al. 2015. Assessment of the allergenic potential of transgenic wheat (Triticum aestivum) with reduced levels of 5-gliadins, the major sensitizing allergen in wheat-dependent exercise-induced anaphylaxis. Journal of Agricultural and Food Chemistry. DOI: 10.1021). Total protein extraction from 100 mg of each of finely pulverized Keumkang, Olgeuru, and Ofree was performed using a 2% SDS sample buffer (2% SDS, 10% glycerol, 0.04 M Tris-HCl, pH 8.5, 50 mM DTT). Isoelectric focusing (IEF) was manually performed using an ampholyte (pI 3-10) and a capillary tube, and the second electrophoresis was performed on a NUPAGE 4-12% Bis-Tris protein gel. Proteins in 2-DE spots were identified by MS/MS. The MS/MS analysis was carried out by the method described in a document (Dupont F M, Vensel W H, Tanaka C K, Hurkman II W J, Altenbach S B. 2011. Deciphering the complexities of the wheat flour proteome using quantitative two-dimensional electrophoresis, three proteases and tandem mass spectrometry. Proteome Science. 9(10). Available at: proteomeci.com). For each spot, three types of proteases (trypsin, chymotrypsin, thermolysin) were used to obtain maximum amino acid sequence coverage. The NCBI protein accession numbers obtained as a result of protein identification within each spot are provided in FIG. 7. After 2-DE, the total protein was transferred to a nitrocellulose membrane. An antibody was prepared using RLLSPRGKELG (SEQ ID NO:36) which is a sequence specific to -5 gliadins and was used for immunoblotting.

(42) All seed storage protein fractions in Keumkang, Olgeuru, Ofree (DH20) and Butte 86 were subjected to two-dimensional electrophoresis and immunoblotting was performed using the serum of a WDEIA patient. The results of protein analysis by MS/MS are provided, in a superimposed manner, along with the immunoblotting results of the WDEIA patient (FIG. 7). An -5 gliadin (BAE20328), i-type LMW-GS (AAS10189), s-type LMW-GSs (BAD12055, AEI00677) and m-type LMW-GS (AEI00671) were identified from Keumkang and -5 gliadins (BAE20328, A1126682), i-type LMW-GSs (AGM38903, ACY08811), s-type LMW-GSs (ACY08813, ACA63868, AEI00677) and m-type LMW-GSs (AGK83389, ACP27643) were identified from Olgeuru. On the other hand, as clearly seen in FIG. 7, no antigen-antibody reaction associated with -5 gliadins was observed in the WDEIA patent. Therefore, it was confirmed that Ofree (DH20) can reduce an allergic immune response leading to WDEIA.

(43) Hence, the inventors of the present invention deposited Ofree with Korean Agricultural Culture Collection (KACC) of National Institute of Agricultural Sciences (166, Nongsaengmyeong-ro, lseo-myeon, Wanju-gun, Jeollabuk-do. Republic of Korea) on Sep. 21, 2015, with the deposit number KACC88001BP.

Example 3: Characterization of Ofree

(44) Various characteristics such as intrinsic characteristics, agricultural traits, yield characteristics, disease resistance, quality characteristics and baking-related characteristics of Ofree developed by the processes described in Example 1 were determined in accordance with Analysis Standards for Research in Agricultural Science and Technology (Rural Development Administration, 2012) and a guideline for characterization of crops in preparation for testing new varieties were provided by the National Seed Management Office of Ministry of Agriculture and Forestry, and the obtained results were compared to the characteristics of Keumkang and Olgeuru, the parent wheat plants.

Example 3-1. Intrinsic Characteristics

(45) The intrinsic characteristics of Ofree in terms of seeding establishment, cold resistance, growth level, leaf color, growth habit, branches and leaves, and uniformity were determined (Table 2).

(46) TABLE-US-00003 TABLE 2 Intrinsic characteristics of Ofree Wheat cultivars Seeding Cold Leaf Growth Branches and and wheat lines establishment resistance Growth color habit leaves Uniformity Keumkang Good 3 Good Light green Semi-erect Slightly drooping Good Olgeuru Good 3 Good Yellow-green Semi-open Slightly drooping Good Ofree Good 3 Good Yellow-green Semi-erect Slightly drooping Good

(47) As shown in Table 2, Ofree had generally similar intrinsic characteristics to those of Keumkang and Olgeuru, except for the leaf color which was similar to that of Olgeuru and the growth habit, which was similar to that of Keumkang.

Example 3-2. Agricultural Traits

(48) The agricultural traits of Ofree in terms of heading date, maturing date, stem length and panicle length were determined (Table 3).

(49) TABLE-US-00004 TABLE 3 Agricultural traits of Ofree Wheat cultivars Heading Maturing Stem length Panicle length and wheat lines date date (cm) (cm) Keumkang May 4th June 11th 69 9.4 Olgeuru May 6th June 13th 66 8.6 Ofree May 9th June 12th 66 9.2

(50) As shown in Table 3, the heading date and maturing date of Ofree were generally similar to those of Keumkang and Olgeuru, and the stem length of Ofree was the same as that of Olgeuru, and the panicle length of Ofree was similar to that of Keumkang.

Example 3-3. Yield Characteristics

(51) The yield characteristics of Ofree in terms of the number of spikes per area, number of grains per spike, thousand-grain-weight, grain weight per liter, and yield per area were determined (Table 4).

(52) TABLE-US-00005 TABLE 4 Yield characteristics of Ofree Number Thousand- Grain of spikes Number grain- weight Yield Wheat cultivars per area of grains weight per liter per area and wheat lines (/m.sup.2) per spike (g) (g) (kg/10a) Keumkang 1,041 35 42.4 806 422 Olgeuru 880 37 41.5 815 485 Ofree 622 36 47.5 810 386

(53) As shown in Table 4, Ofree had a similar number of grains per spike but a significantly lower number of spikes per area compared to Keumkang or Olgeuru, and thus a significantly lower yield per area compared to Keumkang or Olgeuru. However, Ofree had a more desirable yield quality compared to Keumkang or Olgeuru in some aspects by having a larger thousand-grain-weight compared to Keumkang or Olgeuru and an intermediate grain weight per liter between Keumkang and Olgeuru.

Example 3-4. Disease Resistance

(54) To evaluate the disease resistance of Ofree, the resistance of Ofree to Fusarium head blight, virus infection, wheat flour mildew and sheath eyespot was examined (Table 5).

(55) TABLE-US-00006 TABLE 5 Disease resistance of Ofree Wheat Fusarium cultivars head Virus Flour Sheath and blight (high, infection mildew eyespot wheat medium, (high, (high, (affected, not lines low) medium, low) medium, low) affected) Keumkang Medium-low Medium Medium-high Not affected Olgeuru Low Medium-high Medium-high Not affected Ofree Low Medium-high Medium-high Not affected

(56) As shown in Table 5, Ofree had the same level of disease resistance as Olgeuru.

Example 3-5. Baking-Related Characteristics

(57) The baking-related characteristics of Ofree in terms of flour milling characteristics, dough characteristics, and baking characteristics were determined.

Example 3-5-1. Flour Milling Characteristics

(58) The harvested Ofree was milled into flour in a Buhler mill and the flour milling percentage, ash content and flour color of the wheat flour were determined after one month of flour aging (Table 6). In this case, the ash content in the flour was measured in accordance with AACC Method 08-01 (AACC International 2000), and the flour color in terms of whiteness, redness and yellowness was measured with Minolta JS-555 (Konica Minolta, Inc., Japan) and the flour color results were expressed in terms of CIELAB L* (whiteness) a* (redness), and b* (yellowness) axes.

(59) TABLE-US-00007 TABLE 6 Flour milling characteristics of Ofree Flour milling Wheat cultivars percentage Flour color and wheat lines (%) Ash (%) L a b Keumkang 72.57 0.46 91.62 2.10 9.21 Olgeuru 67.19 0.40 92.65 1.53 7.89 Ofree 71.50 0.44 91.55 2.00 8.72

(60) As shown in Table 6, the flour made from Ofree (i.e. Ofree flour) had flour milling percentage, ash content and flour color that are roughly intermediate between those of the Keumkang flour and Olgeuru flour but more similar to those of the Keumkang flour than to those of the Olgeuru flour. The L value of Ofree was lower than that of Keumkang or Olgeuru.

Example 3-5-2. Dough Characteristics

(61) The total protein content, gluten content, and sedimentation value of the Ofree flour produced according to Example 3-5-1 were measured and the characteristics of a dough made of the Ofree flour in terms of an amount of water added, kneading time, and dough stability were measured (Table 7). In this case, the total protein content and gluten content were measured in accordance with AACC Method 46-30 (AACC International 2000); the sedimentation value was determined by the processes of placing 3 g of the flour (based on 14% moisture content) in a 100 ml cylinder, adding 50 ml of a 0.0004% bromophenol blue solution into the cylinder, shaking the cylinder for 15 seconds twice with a two-minute interval, adding 50 ml of a 2% SDS solution containing 12.5% lactic acid into the cylinder and shaking the cylinder for 15 seconds three times with a two-minute interval, leaving the cylinder for 20 minutes, and then measuring the level of the precipitate; and the dough characteristics were determined using a 10 g mixograph (National Mfg. Co., United States) in accordance with AACC Method 54-40A (AACC International 2000).

(62) TABLE-US-00008 TABLE 7 Dough characteristics of Ofree Wheat Sedimen- Dough characteristics cultivars tation Water Kneading and wheat Protein Gluten value added time Stability lines (%) (%) (ml) (%) (min:sec) (mm) Keumkang 15.66 15.15 70.00 67.00 4:00 18.00 Olgeuru 12.35 10.95 48.50 63.00 2:30 13.00 Ofree 14.78 13.60 66.00 67.00 3:15 16.00

(63) As shown in Table 7, the Ofree flour had a protein content, gluten content, sedimentation value and dough characteristics that are roughly intermediate between those of the Keumkang flour and Olgeuru flour but more similar to those of the Keumkang flour than to those of the Olgeuru flour. The Ofree flour had the characteristics of strong wheat flour by requiring the same amount of water to be added to make a dough as the Keumkang flour and, at the same time, characteristics similar to those of medium wheat flour in terms of the kneading time.

Example 3-5-3. Baking Characteristics

(64) Bread was baked using the Ofree flour produced according to Example 3-5-1, and the bread loaf volume and crumb firmness, which are representative of bread characteristics, of the obtained bread were determined (Table 8). In this case, the bread loaf volume was measured using a loaf volumeter (National Mfg. Co., United States) immediately after baking in the oven, and the crumb firmness was determined by cooling the bread at room temperature for two hours, cutting out a crumb with a thickness of 2.0 cm from the central part of the bread loaf, and using a Texture Analyser with a 2.5 cm-diameter plastic plunger at a rate of 1.0 mm/sec at 25% strain with respect to the crumb thickness.

(65) TABLE-US-00009 TABLE 8 Baking characteristics of Ofree Wheat cultivars Bread loaf volume Crumb firmness and wheat lines (ml) (N) Keumkang 900 2.60 Olgeuru 692 5.04 Ofree 793 2.53

(66) As shown in Table 8, the bread produced using the Ofree flour (i.e. Ofree bread) was rated as having a larger volume and greater softness compared to the Olgeuru bread by having a bread loaf volume and crumb firmness that are intermediate between those of Keumkang bread and Olgeuru bread.

(67) FIG. 8 is a set of images for showing Ofree (DH20), Keumkang, and Olgeuru on their heading date (A), and the grains (B) and bread pieces (C) of Ofree (DH20), Keumkang, and Olgeuru.

Production Example: Production of Cookies Using Flour Obtained from Ofree

(68) Ofree cookies were produced in the following manner.

(69) 150 g of butter was gently beaten in a mixer to loosen the butter, 100 g of sugar was added into the mixer, and the contents were homogenized with a whisk for creaming. Then, 120 g of egg was added into the mixer and blended to form a mixture, and mixed wheat flour (100 g of Ofree flour and 300 g of typical soft wheat flour), 30 g of almond powder, 24 g of cocoa were gently blended with the mixture to prepare a base dough.

(70) 50 g of milk was added little by little to the base dough to adjust the consistency of the dough and 20 g of whipped cream was added to the dough, which was then rolled uniformly to a 1 cm thickness and stored in a freezer. In the freezer, egg white and sugar were applied to the surface of the frozen dough, which was then cut into pieces with a size of 1 cm (width)10 cm (length). The pieces were arranged on a baking tray and then baked in a preheated oven for 15 minutes with a top-heat temperature of 190 C. and a bottom-heat temperature of 165 C. to produce Ofree cookies. The baked cookies were cooled at room temperature for about four hours.

(71) It will be understood by those skilled in the art that various changes or modifications may be made to the present invention without departing from the spirit and scope of the present invention as defined by the appended claims.

(72) [Deposition Number]

(73) Depositary name: Korean Agricultural Culture Collection (KACC)

(74) Deposition number: KACC88001BP

(75) Deposited on: Sep. 21, 2015