Mutant rice line 'Samkwang(SA)-flo3' derived from 'Samkwang' with floury endosperm

Abstract

The present invention relates to a novel mutant line derived from Samkwang, "Samkwang(SA)-flo3," which has floury endosperm and is suitable for producing rice flour exclusively for dry milling, and uses thereof. More specifically, the present invention relates to "Samkwang(SA)-flo3," which has floury endosperm suitable for producing rice flour exclusively for dry milling. It also relates to a method for breeding the rice variety, a composition, kit, and method for identifying floury endosperm characteristics of rice progeny lines of the rice variety, and a food composition containing the rice variety, its seeds, or an extract thereof as an active ingredient. "Samkwang(SA)-flo3" provided by the present invention is a mutant line with floury endosperm traits and similar agronomic traits to the original variety, "Samgwang." Because of its floury endosperm traits, it has high dry milling efficiency and is useful as a new ingredient for various processed rice foods.

Description

[Technical field]

[0001] The present invention relates to a mutant rice line "Samkwang(SA)-flo3" derived from "Samgwang" having a novel floury endosperm and uses thereof. More specifically, the present invention relates to "Samkwang(SA)-flo3" having floury endosperm suitable for producing rice flour exclusively for dry milling, a breeding method for the rice variety, a composition, kit and method for identifying floury endosperm characteristics of progeny rice lines of the rice variety, and a food composition containing the rice variety, its seeds or its extract as an active ingredient. [Background technology]

[0002] Traditionally, rice has been the staple food in Korea, but due to changes in the consumption environment, the demand for rice for eating has been declining since the 1980s. Therefore, in order to improve the competitiveness of Korean rice and expand consumption, it is important to develop rice varieties for various uses as well as related processing technologies.

[0003] To increase the added value of the rice processing industry, rice flour should be distributed in large supermarkets and department stores like wheat flour, and rice flour of various grades should be supplied according to the quality standards for each processed product group. However, due to the hardness of rice grains (grain hardness), the most commonly used rice milling method in Korea is wet milling, which involves a water soaking process in which the rice is soaked in water and crushed. However, wet milling has the problem of causing water pollution, as about 500 liters of washing water is generated when producing 100 kg of rice flour. In addition, additional processes such as sterilization and drying are required to distribute rice flour. Therefore, the need for the development of specialized rice flour varieties suitable for dry milling has been pointed out in order to revitalize the rice processing industry and increase consumption.

[0004] Therefore, the Rural Development Administration developed "Suwon No. 542" from the "Namil" mutant line mutated by sodium azide (NaN3), which has floury endosperm with low grain hardness, making it possible to produce high-quality rice flour using only dry milling with a mill (Patent Document 1, Registration Date: October 8, 2012). Subsequent research revealed that the floury endosperm characteristic of "Suwon No. 542" is controlled by a recessive single factor, "flo7", located on chromosome 5. In addition, by comparing the base sequences of "Namil" and "Suwon No. 542" corresponding to "cytosolic pyruvate orthophosphate dikinase protein" (cyOsPPDK), which is presumed to be a gene that specifies floury endosperm, the presence of the G (Namil) → A (Suwon No. 542) SNP in exon 8 was confirmed (Non-Patent Document 1), and a marker to distinguish the SNP was developed (Patent Document 2; Application Date: November 10, 2017), and a patent application for a "novel floury endosperm gene" was filed (Patent Document 3; Application Date: February 25, 2019, Patent Document 4; Application Date: November 9, 2018). In addition, the Rural Development Administration developed new rice varieties, "Kalmi 1" and "Kalmi 2," which contain floury endosperm suitable for rice flour production using dry milling, have reduced pre-harvest germination, excellent disease resistance, and are highly adaptable to late-season cultivation. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Korean Patent No. 10-1190642 [Patent Document 2] Korean Patent Application No. 10-2017-0149236 [Patent Document 3] Korean Patent Application No. 10-2019-0021794 [Patent Document 4] PCT / KR2018 / 013661 [Non-patent literature]

[0006] [Non-Patent Document 1] Heng Wang et al. MGG, 2018 293: 1151-1158 [Non-Patent Document 2] http: / / helix.wustl.edu / dcaps / dcaps / html [Non-Patent Document 3] http: / / primer1.soton.ac.uk / primer1.html [Non-Patent Document 4] Methods 26-10, 26-20, 26-21A, 26-30A, 26-31, 26-41, Approved Methods of the American Association of Cereal Chemists, 10th Edition. 2000. St. Paul, USA [Non-Patent Document 5] Starch Damage in Advances in Cereal Science and Technology. Vol. VII. pp321-349, American Association of Cereal Chemists Inc.St. Paul, USA [Non-Patent Document 6] Approved Methods of the American Association of Cereal Chemists, 10th Edition. 2000. St. Paul, USA Summary of the Invention [Problem to be solved by the invention]

[0007] In this invention, we developed "Samkwang(SA)-flo3", which has a floury endosperm with low grain hardness and can be easily milled, from a group of fixed lines (a total of more than 5,000) of the highest quality rice "Samkwang" mutated with sodium azide, and completed the present invention. [Means for solving the problem]

[0008] The present invention aims to provide a rice cultivar, 'Samkwang(SA)-flo3', a mutant line derived from 'Samkwang' having floury endosperm traits, deposited under Accession No. KACC98101P.

[0009] Another object of the present invention is to provide a method for breeding the rice variety 'Samkwang(SA)-flo3', a progeny line of the 'Samgwang' mutation. Furthermore, the present invention aims to provide a composition for distinguishing floury endosperm characteristics of a progeny line of rice "Samkwang (SA)-flo3", comprising an agent for detecting a single nucleotide polymorphism (SNP) marker at a position corresponding to 19,721,940 bp of chromosome 5 in the rice reference genome (IRGSP 1.0).

[0010] Furthermore, the present invention aims to provide a kit for identifying floury endosperm characteristics of progeny lines of "Samkwang (SA)-flo3", comprising the composition. Furthermore, the present invention aims to provide a method for distinguishing floury endosperm characteristics of a progeny line of rice "Samkwang (SA)-flo3", which comprises a step of detecting a single nucleotide polymorphism (SNP) marker at a position corresponding to 19,725,941 bp of chromosome 5 in the rice reference genome (IRGSP 1.0).

[0011] A further object of the present invention is to provide a food composition containing the rice variety, its seeds, or an extract thereof as an active ingredient. Effect of the Invention

[0012] The "Samkwang(SA)-flo3" provided in the present invention is a mutant line with floury endosperm characteristics and agronomic characteristics similar to those of the original variety "Samgwang." Since it has floury endosperm characteristics, it has high dry milling efficiency and is useful as a new ingredient for various rice processed foods. [Brief description of the drawings]

[0013] [Figure 1a]This is a photograph of the new variety 'Samkwang(SA)-flo3' provided by the present invention and the original variety 'Samgwang', which were grown and reached the yellow ripe stage in the experimental field of the Crop Breeding Division of the National Institute of Food Science and Technology (Wanju-gun, Jeollabuk-do) in 2020. [Figure 1b] 1 is a photograph of the ears of the new variety "Samkwang(SA)-flo3" provided in the present invention and the original variety "Samgwang." [Figure 1c] 1 is a diagram showing the appearance of the new variety of unhulled rice and brown rice provided by the present invention, and shows the cross section of the brown rice and its endosperm to show that it has floury endosperm. [Diagram 2] FIG. 1 shows the results of determining the base sequence at the [G / A] SNP position (19,721,943 bp) of "Samgwang" and "Samkwang(SA)-flo3" confirmed in exon 7 of "Os05g0405000-02," an ORF (Open Reading Frame) containing "cytosolic pyruvate orthophosphate dikinase protein" (cyOsPPDK), which is presumed to be a gene that determines floury endosperm. It was confirmed that the original non-glutinous rice variety "Samgwang" has a [G] base, and "Samkwang(SA)-flo3" provided in the present invention has an [A] base. [Diagram 3] FIG. 2 shows the base sequence and restriction enzyme (RsaI) treatment site of the fragment amplified by the dCAPS primer set according to the present invention. [Figure 4] FIG. 1 shows the results of electrophoresis of "Samkwang" and "Samkwang(SA)-flo3" using the Tetra-Primer ARMS primer set according to the present invention. [Diagram 5] 1 shows the results of electrophoresis of the progeny of the cross between 'Samgwang' and 'Samkwang(SA)-flo3' using the dCAPS primer set and restriction enzyme (RsaI) according to the present invention. In detail, the results are of the phenotype of F2:3 seeds and the genotype of F2, and the homozygote for the allele of 'Samgwang' is indicated as 'A', and the homozygote for the allele of 'Samkwang(SA)-flo3' is indicated as 'B'. [Figure 6] FIG. 1 shows the results of electrophoresis of Korean rice varieties with similar genetic backgrounds using the dCAPS primer set and restriction enzyme (RsaI) according to the present invention (1. Goeun, 2. Ungwang, 3. Chosaeng Bukjal, 4. Odae 1, 5. Shinunbong 1, 6. Manna, 7. Kumoo 3, 8. Hwanggeum Bora, 9. Nungkun Bukjal, 10. Asemi, 11. Ondami, 12. Chungmo 1032, 13 .Jin Ok, 14. Baek Il Mi, 15. Asemi 1, 16. Haedamsal, 17. Mi Hyang Byo, 18. Shin Dong Jin, 19. Su Jin, 20. Ho Jin, 21. Hukhyang, 22. Chunam, 23. Ko Ami, 24. Dong Jin 1, 25. Hyang Mi Byo 1, 26. Nokyang, 27. Segye Jin Mi, 28. Han Areum 3, 29. Han Areum 4, 30. Geum Gang 1, 31. Mok Woo, 32. Mokyang). [Figure 7] 1 shows the results of electrophoresis of the progeny of the cross between 'Samgwang' and 'Samkwang(SA)-flo3' using the Tetra-Primer ARMS primer set according to the present invention. In detail, the results are of the phenotype of F2:3 seeds and the genotype of F2, and the homozygote for the allele of 'Samgwang' is indicated as 'A', and the homozygote for the allele of 'Samkwang(SA)-flo3' is indicated as 'B'. [Figure 8] This is a photograph of a sample that was treated with a commercial kit (Starch Damage Assay Kit, Megazyme) to measure the damaged starch content of rice flour produced by the test mill, and colored to quantify the damaged starch content with a spectrophotometer. All rice flours produced from the six fractionation systems (B1, R1, B2, R2, B3, R3) of the test mill "Buhler MLU-02" were mixed and used. The average damaged starch content measured was 12.0% for "Samkwang", 6.0% for "Samkwang (SA)-flo3", and 4.9% for "Kalmi 2". DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] These will be described in detail below. Note that each description and embodiment disclosed in the present invention is also applicable to other descriptions and embodiments. In other words, all combinations of various elements disclosed in the present invention are included in the present invention. In addition, the present invention is not limited to the following specific description.

[0015] Additionally, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the present invention.

[0016] One aspect of the present invention provides a rice cultivar, 'Samkwang(SA)-flo3', a mutant line derived from 'Samkwang' having floury endosperm trait, deposited under accession number KACC98101P.

[0017] In the present invention, "floury endosperm" refers to a mutation that has a cloudy, opaque appearance like glutinous rice and has irregular starch grain arrangement. Dry and wet milling are used to produce rice flour. Dry milling is a simple process but has the problem of increased damaged starch, while wet milling improves processability but is limited by the time and cost required for soaking and drying. In order to contribute to the revitalization of the rice processing industry, there is a demand for diversification of processing varieties with characteristics suitable for each application and development of varieties with excellent milling properties, and such floury endosperm characteristics are one of the important characteristics that solve the problems of conventional dry milling.

[0018] Rice containing floury endosperm traits is advantageous for dry milling, which allows rice flour production with minimal water pollution, is environmentally friendly, and reduces processing costs. The rice variety according to the present invention is a variety selected as a floury endosperm through objective evaluation from the "Samkwang" mutation fixed line group treated with sodium azide (NaN3). The variety was named "Samkwang(SA)-flo3" and deposited at the National Academy of Agricultural Sciences, Rural Development Administration under the accession number KACC98101P on October 29, 2020.

[0019] The "Samkwang (SA)-flo3" rice variety is a variety that was transferred from domestic deposit to international deposit under the Budapest Treaty at the Korean Academy of Agricultural Sciences (KACC) of the Rural Development Administration on July 5, 2021, with international deposit number KACC88004BP.

[0020] The 'Samkwang(SA)-flo3' rice variety may have at least one of the following characteristics: (a) Heading date: August 14 ± 10 days (b) Rod length (cm): 76±20 (c) Panicle length (cm): 19±10 (d) Number of spikes: 15±5 (e) Weight of 1,000 grains of brown rice (g): 18.5±5.0 (f) Length of brown rice (mm): 5.09 ± 3.00 (g)Brown rice width (mm): 2.97±2.00 (h) Brown rice length-width ratio: 1.72±0.50 (g) Genotype of floury endosperm genes (flo4-6) of “Samkwang(SA)-flo3” (h) The sequence of the SNP in the 7th EXON in the ORF of the floury endosperm gene cyOsPPDK (cytosolic pyruvate orthophosphate dikinase protein) is A (i) Kernel hardness of 3.0 ± 0.34 kg of seeds (j) Heading date of normal cultivation in Wanju on August 14th (k) Average size of seed powder: 65.3 ± 0.86 μm (l) Percentage of damaged starch in 6.0% seed powder Another aspect of the present invention provides a method for breeding the rice variety 'Samkwang(SA)-flo3', a progeny line of the 'Samkwang' mutation.

[0021] The terms used here are as described above. The method may include the steps of (a) soaking "Samgwang" seeds in a sodium azide (NaN3) solution, germinating and cultivating the resulting plant (M1), harvesting M2 seeds from the plant (M1), and cultivating the M2 seeds through pedigree breeding up to the M7 generation to establish fixed mutant lines, and (b) selecting rice varieties having floury endosperm from the seeds of each line established as described above.

[0022] The step (a) may involve soaking "Samgwang" seeds in a sodium azide (NaN3) solution, germinating and cultivating the resulting plant body (M1), harvesting the M2 seeds from the plant body, and cultivating the M2 seeds through pedigree breeding up to the M7 generation to establish a fixed mutant line.

[0023] The concentration of the sodium azide solution in step (a) is not particularly limited to these, but is, for example, 10 to 200 mM, another example, 50 to 150 mM, and yet another example, 100 mM.

[0024] The immersion temperature in the step (a) is not particularly limited to these, but is, for example, 15 to 30°C, and another example is 25°C. The immersion time in step (a) is not particularly limited to these, but is, for example, 1 to 12 hours, another example, 3 to 9 hours, and yet another example, 6 hours.

[0025] The step (b) may involve selecting rice varieties having floury endosperm from seeds of each line established as described above. For example, the (b) step may be carried out by reproducing the seeds of each mutant line established in the (a) step, and culturing the mutant line "Suwon No. 542 ( powderThe method involves visually selecting lines that express endosperm similar to the "quality of the endosperm."

[0026] In the present invention, the steps (a) and (b) may be appropriately modified depending on the changes in the floury endosperm, the characteristics of the mutant line, and the like. Yet another aspect of the present invention provides a composition for identifying floury endosperm characteristics of a progeny line of rice "Samkwang (SA)-flo3," comprising an agent for detecting a single nucleotide polymorphism (SNP) marker at a position corresponding to 19,721,940 bp of chromosome 5 in the rice reference genome (IRGSP 1.0).

[0027] The terms used here are as described above. In the present invention, a "single nucleotide polymorphism" (SNP) refers to a gene locus in which two or more alleles exist, and a polymorphism in which only one nucleotide differs at the polymorphic site is called a single nucleotide polymorphism. The term "allele" refers to various forms of a gene that exist at the same locus of a homologous chromosome, and an SNP has two alleles.

[0028] The SNP in the present invention is a polynucleotide sequence (ch; 19,718,538-19,726,410) derived from "Samkwang (SA)-flo3" represented by SEQ ID NO: 1 located on chromosome 5. 333 3, 333 This means that the G at the 354th position has been replaced with A, and therefore the 354th amino acid glycine (Gly, G) has been mutated to aspartic acid (Asp, D).

[0029] In the present invention, the preparation for detecting the SNP marker comprises a polynucleotide sequence represented by SEQ ID NO: 1, 333The primer may be a primer that amplifies a polynucleotide consisting of 10 to 300 bases and containing a single nucleotide polymorphism site, which is located at position 1 of the sequence, or a probe that specifically hybridizes to the polynucleotide.

[0030] Specifically, the preparation for detecting the SNP marker is a primer set consisting of the primers shown in SEQ ID NO: 4 to SEQ ID NO: 7, but is not limited thereto. In the present invention, the term "primer" refers to a short sequence having a free 3' hydroxyl group at its short 3' end, capable of forming a base pair with a complementary template, and functioning as a starting point for copying the template. The appropriate length of a primer varies depending on the intended use, but generally consists of 15 to 30 bases. The primer sequence does not need to be completely complementary to the template, but must be sufficiently complementary to be able to hybridize to the template. The primer can amplify a DNA fragment containing the polymorphic site by hybridizing to a DNA sequence containing the polymorphic site. For the purposes of the present invention, the primer is powder The primer set amplifies a single nucleotide polymorphism site closely associated with quality endosperm characteristics, and is, for example, a primer set consisting of primers represented by SEQ ID NO: 4 to SEQ ID NO: 7, but is not limited to this.

[0031] The primer set consists of any base sequence having preferably 80% or more sequence homology, more preferably 90% or more sequence homology, even more preferably 95% or more sequence homology, and most preferably 99% or more sequence homology to each of the base sequences represented by SEQ ID NO: 4 to SEQ ID NO: 7, but any primer may be used as long as it is capable of amplifying the SNP site described above.

[0032] The primer or probe of the present invention can be chemically synthesized using phosphoramidite solid support method or other known methods. Such nucleic acid sequences can also be modified using many means known in the art. Examples of such modifications include, but are not limited to, methylation, capping, substitution of at least one of the natural nucleotides with its analogue, and internucleotide modifications, such as uncharged linkers (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.) or charged linkers (e.g., phosphorothioates, phosphorodithioates, etc.).

[0033] The present invention provides a kit for identifying floury endosperm characteristics of a progeny line of rice "Samkwang (SA)-flo3", comprising the composition. The terms used here are as described above.

[0034] In addition to the above-mentioned composition, the kit according to the present invention may further include a DNA polymerase, dNTPs, and a PCR buffer solution for facilitating the PCR reaction, as well as components necessary for performing electrophoresis to confirm whether the PCR product has been amplified, or an identification reference table for known varieties.

[0035] Yet another aspect of the present invention provides a method for identifying floury endosperm characteristics of a progeny line of rice "Samkwang (SA)-flo3", the method comprising the step of detecting a SNP marker at a position corresponding to 19,725,941 bp of chromosome 5 in the rice reference genome (IRGSP 1.0).

[0036] The terms used here are as described above. The base of the SNP marker at a position corresponding to 19,721,940 bp of chromosome 5 in the rice reference genome (IRGSP 1.0) may be A.

[0037] The step of detecting the SNP includes, but is not limited to, (a) a step of performing PCR using genomic DNA isolated from a sample as a template and a primer set consisting of primers represented by SEQ ID NO: 4 to SEQ ID NO: 7, and (b) a step of confirming the product amplified by the PCR.

[0038] Yet another aspect of the present invention provides a food composition comprising the rice variety, its seeds or an extract thereof as an active ingredient. The terms used here are as described above.

[0039] Specifically, the rice variety is "Samkwang (SA)-flo3," as described above. The seeds of the variety of the present invention can be dry milled to produce high-quality rice flour with fine particles and little damage to the starch granules, so food compositions containing this as an active ingredient are price competitive and have an excellent texture.

[0040] The food composition of the present invention may comprise rice flour produced from the seeds of said rice varieties. The rice flour may be produced by dry milling.

[0041] The food composition of the present invention includes all forms of rice processed food, functional food, nutritional supplement, health functional food, food additives, etc. The above-mentioned types of food compositions can be prepared in various forms by conventional methods known in the art.

[0042] For example, rice processed foods such as rice cakes and noodles (rice cakes, tteokbokki rice cakes, udon noodles, ramen noodles, buckwheat noodles, fresh noodles, traditional rice cakes), rice snacks (biscuits, dry bread, snacks, Korean sweets, popped rice cakes, scorched rice), rice flour (fresh rice flour, alpha rice flour, wet rice flour), alcoholic beverages (cloudy wine, shochu, beer, sake), seasonings (candy, soy sauce, vinegar) and other rice processed products (porridge, sikhye, snack secondary ingredients, skewers, Zen foods, rice beverages, rice flour bread, processed rice) can be produced by processing the rice variety provided in the present invention or its seeds.

[0043] In the present invention, the term "functional food" is synonymous with food for special health use (FoSHU), and refers to food with high medical and therapeutic effects that is processed to efficiently exhibit bioregulatory functions in addition to providing nutrition. Here, "functionality" refers to regulating nutrients to the structure and functions of the human body, or providing beneficial effects for health purposes such as physiological actions.

[0044] The ingredients other than the "Samkwang(SA)-flo3" rice variety, its seeds or its extracts, which are essential ingredients contained in the food composition of the present invention, are not particularly limited, and may contain various herbal extracts, food supplement additives, natural carbohydrates, etc. as additional ingredients, similar to ordinary foods. In addition, the food supplement additives include food supplement additives commonly used in the art, such as flavoring agents, flavoring agents, coloring agents, fillers, stabilizers, etc.

[0045] The food of the present invention can be produced by a method commonly used in the art, and can be produced by adding raw materials and ingredients commonly added in the art. The dosage form of the food may be any dosage form recognized as a food. The food composition of the present invention can be produced in various dosage forms, and unlike general medicines, it has the advantage of being free of side effects that may occur when taking medicines for a long period of time, as it uses food as a raw material, and is highly portable.

[0046] The content of the plant, its seeds or its extract contained in the food composition of the present invention is, for example, 0.01 to 95% by weight of the final food product, but is not limited to this. EXAMPLES

[0047] The present invention will be described in more detail below with reference to examples and experimental examples. However, these examples and experimental examples are merely illustrative of the present invention, and the present invention is not limited to these examples and experimental examples. EXAMPLES

[0048] Selection of novel floury endosperm rice varieties Example 1-1: Establishment of the 'Samgwang' mutant fixed lineage Mutant progeny lines were obtained by treating seeds of the high-quality Korean rice (Oryza sativa L.) variety "Samgwang" with sodium azide (NaN3) diluted in potassium phosphate buffer as a mutagen. One ear was harvested from the M1 plant (M2 seed), and then the generations were advanced to the M7 generation by pedigree breeding to establish fixed lines. One highly fertile plant was randomly selected from each line at each generation, and progeny lines were developed using the one ear, one row method. At the M7 plant generation, individuals within each line showed a uniform phenotype, so it was determined that the lines were genetically fixed, and a total of more than 5,000 lines were confirmed. EXAMPLES

[0049] Agronomic characterization The 'Samkwang (SA) -flo3' variety was tested in the National Institute of Food Science and Technology's Wanju experimental field under normal season top dressing conditions, and major agronomic characteristics such as heading time, culm length, and panicle length were observed.

[0050] As shown in Table 1 and Figures 1a and 1b, the agronomic characteristics of the 'Samkwang(SA)-flo3' variety selected in Example 1 and the original variety ('Samgwang') were compared. As a result, it was confirmed that the 'Samkwang(SA)-flo3' variety has smaller grain hardness, number of grains per ear, and thousand-kernel weight of brown rice compared to 'Samgwang', but the agronomic characteristics such as heading time, culm length, ear length, and number of ears are at the same level as the mid-late maturing original variety ('Samgwang').

[0051] In addition, on November 8, 2020, 'Samkwang(SA)-flo3' was determined to be suitable for the selection requirements for varieties specifically for mid-late maturing rice flour, and was selected as 'Chongju No. 672', an excellent test line for the 2021 new regional rice adaptation test.

[0052] [Table 1]

[0053] Therefore, the "Samkwang(SA)-flo3" was deposited at the Korea Agricultural Microbial Resource Center, National Institute of Agricultural Sciences, Rural Development Administration, on October 29, 2020 under the accession number KACC98101P. EXAMPLES

[0054] Selection of rice varieties with floury endosperm The appearance of brown rice was evaluated for the 'Samgwang' mutant fixed group established in Example 1-1, and a line with whitish floury endosperm was selected. As shown in Figure 1c, floury endosperm and Ulchi rice could be distinguished from each other with a clear view.

[0055] Example 3-1: Floury endosperm F 2:3 DNA extraction and phenotypic evaluation of populations for genetic analysis For the genetic analysis of floury endosperm, F2 populations, which were grown by self-fertilizing F1 plants derived from the cross between the original varieties 'Samgwang' and 'Samkwang(SA)-flo3', were tested at the National Institute of Food Science and Technology greenhouse in June 2020. The results of the objective evaluation of brown rice of the F2 seeds of the cross progeny of 'Samgwang' and 'Samkwang(SA)-flo3' showed 1,122 non-glutinous rice and 363 floury endosperm, with a segregation ratio of 3:1. From the expanded F2 population, a total of 419 individuals, consisting of 338 non-glutinous rice and 81 floury endosperm, were randomly selected and determined as the genetic analysis population to explore the gene loci controlling the floury endosperm characteristics of 'Samkwang(SA)-flo3'.

[0056] Leaf samples were taken from the genetic analysis population, Samkwang(SA)-flo3, and the original variety Samkwang, and DNA was extracted using the modified CTAB method (Murray and Thompson, 1980). The extracted DNA was electrophoresed on a 1% agarose gel, quantified using a NanoDrop spectrophotometer (Thermo Fisher Scientific, USA), and then diluted to the appropriate concentration for PCR reaction. EXAMPLES

[0057] Elucidation of single nucleotide polymorphism sites associated with floury endosperm characteristics of "Samkwang(SA)-flo3" Example 4-1: DNA extraction DNA from "Samkwang" and "Samkwang(SA)-flo3" with floury endosperm characteristics was extracted from young leaves by the CTAB method (Murray & Thomason, 1980). The extracted DNA was confirmed by 0.8% agarose gel electrophoresis and quantified using a NanoDrop spectrophotometer (Thermo Fisher Scientific, USA), and then diluted to 5ng / μl for PCR.

[0058] Example 4-2: Comparison of PPDK1 (OS05G045000-02) gene sequences between "Samkwang" and "Samkwang(SA)-flo3" Based on the rice reference genome (IRGSP 1.0) information, the transcriptome information of the candidate gene PPDK1 (pyruvate, phosphate dikinase 1) located on chromosome 5 was confirmed, and the base sequence of 19,718,538-19,726,410 (7,873 bp) in PPDK1 of OS05G0405000-02 (19,718,538-19,737,605 bp) was analyzed (SEQ ID NO: 1, Accession No.: MW349590). As shown in Figure 2, the SNP associated with floury endosperm characteristics was confirmed to be "G" in Samkwang and "A" in Samkwang(SA)-flo3 at the position corresponding to 19,721,940 bp of the IRGSP reference genome. More specifically, it was confirmed that the 354th amino acid of OS05G0405000-02 was mutated from glycine (Gly, G) to aspartic acid (Asp, D). EXAMPLES

[0059] Preparation of primer set for distinguishing floury endosperm characteristics Example 5-1: Preparation of dCAPS primer set dCAPS primers were prepared using dCAPS Finder 2.0 (Non-Patent Document 2) for the SNPs elucidated in Example 4. For the region amplified by dCAPS, a 21-bp overhang was added to 19,718,217,67-19,721,944 bp (197 bp) of chromosome 5, which contains the SNP (19,721,940 bp) determined to be polymorphic, to amplify the total 218 bp, and a 1-bp mismatch primer was searched for.

[0060] [Table 2]

[0061] PCR was performed in a total volume of 20 ul using 10 ng of DNA, 5 pmol of forward and reverse primers, 0.2 mM dNTP mix, 1X PCR buffer [50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton® X-100, 1.5 mM MgCl2], and 1 unit of Taq polymerase. After initial denaturation at 95°C for 5 minutes, 40 cycles of 95°C for 20 seconds, 58°C for 30 seconds, and 72°C for 60 seconds were repeated, followed by reaction at 72°C for 10 minutes.

[0062] The restriction enzyme used was RsaI, which cleaves the "5...GTAC...3" site. As shown in Figure 3, "Samkwang" has a GTAC sequence due to SNP, so it was designed to be cleaved by the restriction enzyme, while "Samkwang(SA)-flo3" was designed not to be cleaved by the restriction enzyme. Restriction enzyme treatment was performed in a total volume of 15 uL using 1X enzyme buffer [50 mM potassium acetate, 20 mM Tris acetate, 10 mM magnesium acetate, 100 ug / ml BSA], 10 ng of PCR product, and 5U RsaI (NEB, UK). The products were electrophoresed on an agarose gel, and then the genotype was determined.

[0063] As a result, as shown in Table 3 and Figure 3, "Samkwang" and "Samkwang(SA)-flo3" were amplified at 218 bp, but it was confirmed that "Samkwang" was cleaved at 175 bp and 43 bp by the RsaI restriction enzyme, while "Samkwang(SA)-flo3" was not cleaved.

[0064] [Table 3]

[0065] Example 5-2: Preparation of Tetra-primer ARMS primer set In order to prepare primers that can omit the restriction enzyme treatment step for the searched SNPs, Tetra-Primer ARMS-PCR primers were developed using the Primer1 (Primer Design for Tetra-Primer ARMS-PCR, Non-Patent Document 3) service. As shown in Table 4, a primer set was selected that can distinguish the SNPs of "Samkwang(SA)-flo3", which has floury endosperm characteristics, without treatment with restriction enzymes.

[0066] DNA from "Samkwang" and "Samkwang(SA)-flo3" with floury endosperm characteristics was extracted from young leaves by the CTAB (Cetyltrimethylammonium bromide) method (Murray & Thomason, 1980). The extracted DNA was confirmed by 0.8% agarose gel electrophoresis and quantified using a NanoDrop spectrophotometer (Thermo Fisher Scientific, USA), and then diluted to 5ng / μl for PCR.

[0067] [Table 4]

[0068] Specifically, when PCR was performed using the Flo4-6 Tetra-Primer ARMS-PCR primer, it was revealed that "Samkwang", which has the G allele, has a 644 bp PCR fragment due to Flo4-6-outer-F and Flo4-6-outer-R, and a 377 bp fragment due to Flo4-6-inner-F(G) and Flo4-6-outer-R binding to the G allele, whereas "Samkwang(SA)-flo3", which has the A allele, has a 644 bp fragment due to Flo4-6-outer-F and Flo4-6-outer-R, and a 308 bp fragment due to Flo4-6-inner-R(A) and Flo4-6-outer-F binding to the A allele.

[0069] [Table 5]

[0070] PCR was performed in a total volume of 20 ul using 10 ng of DNA, 5 pmol of each of the four primers, 0.2 mM dNTP mix, 1X PCR buffer [50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton® X-100, 1.5 mM McCl2], and 1 unit of Taq polymerase. After initial denaturation at 95°C for 5 min, the cycle of 95°C for 20 s, 59°C for 30 s, and 72°C for 60 s was repeated 35 times, followed by reaction at 72°C for 10 min. The products were electrophoresed on a 4% agarose gel and then genotyped.

[0071] As a result, as shown in Figure 4, a specific fragment of 'Samkwang(SA)-flo3' was obtained by PCR using the Flo4-6 Tetra-Primer ARMS-PCR primers prepared as described above. In other words, it was found that the floury endosperm characteristics of 'Samkwang(SA)-flo3' can be effectively distinguished by these differences. EXAMPLES

[0072] Verification of the effectiveness of primers for distinguishing floury endosperm characteristics of "Samkwang(SA)-flo3" Example 6-1: 'Samkwang' x 'Samkwang(SA)-flo3' F 2:3 Verification of the effectiveness of dCAPS primer set Progeny F of the cross between 'Samkwang' and 'Samkwang(SA)-flo3' 2:3 419 plant bodies were analyzed under the same conditions as in Example 5, using "Samgwang" and "Samkwang(SA)-flo3" as the female and male plants. If the form that is cleaved at 175 bp and 43 bp by the restriction enzyme RsaI is defined as type A, and the form that is not cleaved by the restriction enzyme RsaI is defined as type B, then "Samgwang" was type A, and "Samkwang(SA)-flo3", which has floury endosperm, was type B. As shown in FIG. 5, the progeny F 2:3The genotype of 338 plants of Uruchi rice was heterozygous for A type, and F 2:3 Since the genotypes of all 81 floury endosperm plants were type B, it was confirmed that the phenotype of non-glutinous rice was dominant to that of floury endosperm. powder It was found that it can be effectively applied to quality trait discrimination.

[0073] Example 6-2: Verification of the effectiveness of dCAPS primer set for Korean rice varieties In addition, to confirm the effect of selection in a population with a similar genetic background, DNA from 32 Korean rice cultivars bred in Korea was used (1. Goeun, 2. Ungwang, 3. Chosaeng Bukjal, 4. Odae 1, 5. Shinunbong 1, 6. Manna, 7. Kumoo 3, 8. Hwanggeum Bora, 9. Nungkun Bukjal, 10. Asemi, 11. Ondami, 12. Chungmo 1032, 13. Jinok, 14. Baekui). Rumi, 15. Asemi 1, 16. Haedamsal, 17. Mi Hyangbyo, 18. Shin Dongjin, 19. Sujin, 20. Hojin, 21. Hukhyang, 22. Chunam, 23. Koami, 24. Dongjin 1, 25. Hyangmibyo 1, 26. Nokyang, 27. Segyejinmi, 28. Hanalum 3, 29. Hanalum 4, 30. Geumgang 1, 31. Mokwoo, 32. Mokyang) were analyzed under the same conditions as in Example 5.

[0074] As a result, as shown in FIG. 6, only “Samkwang(SA)-flo3”, which exhibited floury endosperm characteristics, was type B, and the rest were all type A. From these results, the SNP and dCAPS primers according to the present invention powder It was found that this can be effectively applied to distinguish quality traits. In addition, it was confirmed that the SNP determining floury endosperm is different from that of 'Suwon No. 542' (floury endosperm, flo7), and thus can be distinguished.

[0075] Example 6-3: Verification of the effectiveness of the Tetra-primer ARMS primer set Progeny F of the cross between 'Samkwang' and 'Samkwang(SA)-flo3' 2:3419 plant bodies were analyzed under the same conditions as in Example 5, using "Samgwang" and "Samkwang(SA)-flo3" as the female and male plants. As a result, when the form cleaved at 377 bp by Tetra-primer ARMS PCR was defined as type A and the form cleaved at 308 bp as type B, "Samgwang" was type A and "Samkwang(SA)-flo3", which has floury endosperm, was type B. As a result, as can be seen in Figure 7, the cross progeny F 2:3 The genotype of 338 plants of Uruchi rice was heterozygous for A type, and F 2:3 Since the genotypes of all 81 floury endosperm plants were type B, it was confirmed that the phenotype of non-glutinous rice was dominant to that of floury endosperm. From these results, it was found that the SNP and Tetra-primer ARMS primers according to the present invention are powder It was found that it can be effectively applied to quality trait discrimination. EXAMPLES

[0076] Analysis of physicochemical properties of rice flour Example 7-1: Kernel Hardness Analysis To compare whether the characteristics of floury endosperm, which has low grain hardness and easily breaks down when pressure is applied, are well expressed in the 'Samkwang(SA)-flo3' variety, grain hardness was measured using a TA.XTplus texture analyzer (Satble Micro Systems Ltds. UK) in comparison with the control group. Here, pressure was applied to brown rice using a probe with a diameter of 5 mm (text speed: 0.4 mm / sec, trigger force: 40.0 g), and the pressure at which the sample broke was measured. Each sample was repeated 50 times, and the average and standard deviation were calculated.

[0077] As a result, as shown in Table 6, it was confirmed that the grain hardness of 'Samkwang(SA)-flo3' (3.0 kg) was very low for non-glutinous rice such as the original variety 'Samkwang' (9.2 kg), and was lower than that of the conventional variety exclusively used for dry milling 'Kalmi 2' (3.1 kg).

[0078] [Table 6]

[0079] Example 7-2: Rice flour production using a dry mill Considering the characteristics of the mill in which the seed coat falls off (naked hull), brown rice was used for all rice samples. Dry milling of Samkwang (SA)-flo3, Samkwang (original variety) and control (Kalmi 2) was performed under the same conditions using a laboratory-scale dry mill, Buhler MLU-202 (Buhler Bros. Inc. Swiss). The Buhler mill consists of three sets of break rolls (B1, B2, B3) and reduction rolls (R1, R2, R3). Each roll is connected to a trommel process, and by-products (Bran; bran, Short; powder) and six flour fractions from each fractionation system (B1, R1, B2, R2, B3, R3) are ultimately produced. The procedure for dry milling using a Buhler MLU-202 was as described in reference (Non-Patent Document 4). In the present invention, all the rice flours produced from the six fractions were mixed and evaluated against rice flour produced by dry milling.

[0080] Example 7-3: Analysis of grain hardness and physicochemical properties of rice flour A Laser Diffraction Particle Size Analyzer (model LS 13 320, Beckman Mill, Inc.) was used to measure the average size and particle size distribution (diameter distribution) of rice flour produced by dry milling under the same conditions.

[0081] As a result, as shown in Table 7, it was confirmed that the average size of the rice flour of "Samkwang(SA)-flo3" (65.3 μm) was slightly larger than that of the conventional "Karmii 2" (61.5 μm), but was very small compared to that of the original variety "Samgwang" (91.1 μm).

[0082] This trend was also clearly reflected in the average particle size of each size fraction of rice flour, with 'Samkwang(SA)-flo3' being reconfirmed to be crushed finer than other rice varieties, next to the conventional 'Kalmi2' level, even with dry milling alone.

[0083] [Table 7]

[0084] Example 7-4: Measurement of the percentage of damaged starch in rice flour The biggest problem when producing rice flour by dry milling ordinary non-glutinous rice is physical damage to starch granules due to the high kernel hardness of rice. Damage to starch granules has a significant effect on the physical properties of the paste. Damaged starch granules are also known to be rapidly hydrated and easily hydrolyzed by α- or β-amylase and converted into fermentable sugars, which promotes excessive fermentation and leads to inappropriate gas generation during the processing process, resulting in a reduction in the volume of processed products (Non-Patent Document 5).

[0085] Therefore, to evaluate the proportion of damaged starch in rice flour produced by dry milling under the same conditions, a commercially available analysis kit (Starch Damage Assay Kit, Megazyme, Ireland) was used. This kit involves placing milled rice flour in an enzyme reaction solution for reaction, which then develops color and the amount of damaged starch granules is analyzed using a spectrophotometer.

[0086] As a result, as shown in Figure 8, the percentage of damaged starch in rice flour in "Samkwang(SA)-flo3" (6.0%) was not significantly different from that of "Kalmi2" (4.9%), and was significantly lower than that of the original non-glutinous rice variety "Samgwang" (12.0%).

[0087] These results confirmed that the present invention's "Samkwang(SA)-flo3" has floury endosperm with physical properties roughly equivalent to those of "Kalmi2" and is a rice line capable of producing fine, high-quality rice flour with low damaged starch content using only dry milling.

[0088] Example 7-5: Evaluation of other physicochemical properties of rice flour As a means to understand the phenotype of the rice flour of the present invention "Samkwang (SA)-flo3" produced by the mill, the rice flour lightness (CIE value) was measured under specific moisture content conditions, and the ash content of the rice flour was measured. The investigation and measurement method used here are based on the reference (Non-Patent Document 6).

[0089] As can be seen in Table 6, the rice flour whiteness of the present invention "Samkwang (SA)-flo3" is The rating was 92.6, lower than "Samgwang" (93.2) and higher than "Galmi 2" (91.2). In addition, the ash content, which is generally known to have a high positive correlation with the rate of bran contamination during the flour milling process, was 0.67 for "Samkwang," 0.64 for "Samkwang(SA)-flo3," and 0.58 for "Kalmi2," showing no significant difference.

[0090] From the above description, a person skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing its technical ideas or essential features. It should be understood that the above examples are merely illustrative and not limiting. The present invention should be interpreted as including all modifications and alterations derived from the meaning and scope of the claims and their equivalent concepts, rather than the specification.

[0091] [Table 8]

[0092] [Table 9]

Claims

1. The rice variety "Samkwang (SA)-flo3", a mutant line derived from "Samgwang" having floury endosperm traits, deposited under international accession number KACC88004BP.

2. The rice variety according to claim 1, wherein the rice variety has the following characteristics: (a) Heading date: August 14 ± 10 days (b) Rod length (cm): 76±20 (c) Panicle length (cm): 19±10 (d) Number of ears (pieces): 15±5 (e) Weight of 1,000 grains of brown rice (g): 18.5±5.0 (f) Brown rice length (mm): 5.09 ± 3.00 (g) Brown rice width (mm): 2.97 ± 2.00 (h) Brown rice length-width ratio: 1.72 ± 0.50

3. (a) "Samgwang" seeds were soaked in sodium azide (NaN 3 ) solution, germinate and cultivate the resulting plant (M1), and then harvest M2 seeds from the plant (M1) through pedigree breeding up to the M7 generation to establish fixed mutant lines; (b) selecting rice varieties having floury endosperm from the seeds of each line established as described above, the rice variety being a progeny line of the Samkwang mutation, 'Samkwang (SA)-flo3'.

4. The method according to claim 3, wherein the concentration of the diluted sodium azide solution in step (a) is 10 to 200 mM.

5. A composition for distinguishing floury endosperm characteristics of a progeny line of rice "Samkwang (SA)-flo3," comprising a preparation for detecting a single nucleotide polymorphism (SNP) marker at a position corresponding to the 3,333rd position of a polynucleotide sequence represented by SEQ ID NO:1 on chromosome 5 in the rice standard genome (IRGSP 1.0), wherein the SNP marker at a position corresponding to the 3,333rd position of a polynucleotide sequence represented by SEQ ID NO:1 on chromosome 5 in the rice standard genome (IRGSP 1.0) has an A base.

6. The composition for determining the characteristics of floury endosperm according to claim 5, wherein the preparation for detecting the SNP marker is a primer for amplifying a polynucleotide consisting of 10 to 300 bases, including a single nucleotide polymorphism site present at the 3,333rd position of the polynucleotide sequence represented by SEQ ID NO:1, or a probe for specifically hybridizing to the polynucleotide.

7. The composition for determining floury endosperm characteristics according to claim 6, wherein the preparation for detecting the SNP marker is a primer set consisting of primers represented by SEQ ID NO: 4 to SEQ ID NO:

7.

8. A kit for determining floury endosperm characteristics of a progeny line of "Samkwang (SA)-flo3" rice, comprising the composition according to any one of claims 5 to 7.

9. A method for distinguishing floury endosperm characteristics of a progeny line of rice "Samkwang (SA)-flo3," comprising: detecting a SNP marker at a position corresponding to the 3,333rd position of a polynucleotide sequence represented by SEQ ID NO:1 on chromosome 5 in the rice standard genome (IRGSP 1.0), wherein the SNP marker at a position corresponding to the 3,333rd position of a polynucleotide sequence represented by SEQ ID NO:1 on chromosome 5 in the rice standard genome (IRGSP 1.0) has an A base.

10. The step of detecting the SNP markers comprises: (a) performing PCR using genomic DNA isolated from a sample as a template and a primer set consisting of primers represented by SEQ ID NO: 4 to SEQ ID NO: 7; (b) confirming the PCR amplified product.

11. A food composition comprising the rice variety, its seeds or its extract according to claim 1 or 2 as an active ingredient.

12. 12. The food composition of claim 11, wherein the food product comprises rice flour produced from seeds of the rice variety.

13. 13. The food composition of claim 12, wherein the rice flour is produced by dry milling.

14. A mutant rice line derived from 'Samgwang' having floury endosperm characteristics, comprising a polynucleotide sequence represented by SEQ ID NO:1, wherein position 3,333 in SEQ ID NO:1 is A.

15. A polynucleotide represented by SEQ ID NO:1, wherein position 3,333 is substituted with A.

Images