Method for predicting cooked rice taste of rice of plant of genus oryza

Abstract

To provide a method for predicting cooked rice taste of rice of a plant of the genus Oryza.SOLUTION: The method for predicting cooked rice taste of rice of a plant of the genus Oryza includes evaluating at least one selected from the group consisting of the following (A) to (C): (A) the expression level of mRNA comprising the nucleotide sequence shown in SEQ ID NO: 1 in rice of a plant of the genus Oryza; (B) an expression level of a protein comprising the amino acid sequence shown in SEQ ID NO: 2 in rice of a plant of the genus Oryza; and (C) a nucleotide corresponding to thymine at position 1463 in the region of the genomic DNA of Nipponbare whose nucleotide sequence is shown in SEQ ID NO: 3, in the genomic DNA of a plant of the genus Oryza.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for predicting the eating quality of cooked rice from plants of the genus Oryza. [Background technology]

[0002] Rice is the staple food of the Japanese people. Many varieties of rice are cultivated in Japan, but Koshihikari has the largest planting area, accounting for approximately 36% of the total planted area in Japan. Other varieties with large planting areas besides Koshihikari include Hitomebore, Hinohikari, Akitakomachi, Nanatsuboshi, and Haenuki, all of which are first- or second-generation hybrid offspring of Koshihikari. The reason Koshihikari is the most widely cultivated variety in Japan is because the cooked rice is delicious, meaning it has an excellent cooked rice flavor.

[0003] Many studies have been conducted to date to identify the QTLs (Quantitative Trait Loci) that contribute to the excellent cooked rice taste of Koshihikari, with the main aim of using them as indicators for variety improvement, etc. For example, Non-Patent Document 1 describes that a progeny line in which an approximately 11.28 Mbp region on the short arm of chromosome 3 of Nipponbare was replaced with a Koshihikari-type allele exhibits excellent cooked rice taste. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Yoshinobu Takeuchi et al., "Major QTLs for eating quality of an elite Japanese rice cultivar, Koshihikari, on the short arm of chromosome 3", Breeding Science 58(4), 437-445(2008). Summary of the Invention [Problem to be solved by the invention]

[0005] As mentioned above, the QTL candidate region described in Non-Patent Document 1 covers an extremely wide range of approximately 11.28 Mbp, and the gene directly responsible for the excellent cooked rice taste of Koshihikari has not yet been identified.

[0006] An object of the present disclosure is to provide a method for predicting the eating quality of cooked rice from plants of the genus Oryza. Another object of the present disclosure is to provide a plant of the genus Oryza that exhibits excellent cooked rice taste, a method for producing the same, and a method for improving the eating quality of cooked rice from plants of the genus Oryza. Another object of the present disclosure is to provide a primer set for predicting the eating quality of cooked rice from plants of the genus Oryza. [Means for solving the problem]

[0007] The present inventors have discovered that a single nucleotide polymorphism that causes the insertion of a cis-element in the promoter region of Koshihikari and Nipponbare is present on the short arm of chromosome 3. Furthermore, the present inventors have discovered that the gene downstream of the promoter region where the insertion of the cis-element was caused is a QTL responsible for the excellent eating quality of cooked rice in Koshihikari, and that the expression level of this gene has a significant effect on the eating quality of cooked rice in Oryza plants, thereby completing the present invention.

[0008] The present disclosure relates, for example, to the following: [1] A method for predicting the eating quality of cooked rice from a plant of the genus Oryza, comprising evaluating at least one selected from the group consisting of the following (A) to (C): (A) Expression level of mRNA containing the nucleotide sequence shown in SEQ ID NO: 1 in rice, a plant of the genus Oryza; (B) Expression level of a protein containing the amino acid sequence shown in SEQ ID NO: 2 in rice of the genus Oryza; (C) The base corresponding to the 1463rd thymine in the region of the genomic DNA of the Nipponbare variety whose base sequence is shown in SEQ ID NO: 3 in the genomic DNA of the Oryza plant. [2] The method includes evaluating (A) the expression level of mRNA comprising the nucleotide sequence shown in SEQ ID NO: 1 in rice, a plant of the genus Oryza, and / or (B) the expression level of protein comprising the amino acid sequence shown in SEQ ID NO: 2 in rice, a plant of the genus Oryza; The method described in [1] further comprises predicting that the rice plant will have excellent cooked rice taste if the expression level of the mRNA and / or the protein in the evaluated rice of the rice plant is 1.3 to 6.0 times the expression level of the mRNA containing the base sequence shown in SEQ ID NO: 1 and / or the protein containing the amino acid sequence shown in SEQ ID NO: 2 in Nipponbare rice. [3] The method includes (C) evaluating a base corresponding to thymine at position 1463 in the region of the genomic DNA of the plant of the genus Oryza, the nucleotide sequence of which is set forth in SEQ ID NO: 3 in the genomic DNA of Nipponbare; The method according to [1] or [2], further comprising predicting that the rice plant will have excellent cooked rice taste if the evaluated base corresponding to the 1463rd thymine is cytosine. [4] A method for producing a rice plant having excellent cooked rice taste, the method comprising at least one method selected from the group consisting of the following (a) to (c): (a) increasing the expression level of mRNA containing a nucleotide sequence having 90% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 1 in rice of the genus Oryza to between 1.3 and 6.0 times the expression level of mRNA containing the nucleotide sequence shown in SEQ ID NO: 1 in Nipponbare rice; (b) increasing the expression level of a protein containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 2 in rice of the genus Oryza to between 1.3 and 6.0 times the expression level of a protein containing the amino acid sequence set forth in SEQ ID NO: 2 in Nipponbare rice; (c) In the genomic DNA of a plant of the genus Oryza, the base corresponding to thymine at position 1463 in the region of the genomic DNA of Nipponbare, whose base sequence is set forth in SEQ ID NO: 3, is replaced with cytosine. [5] A method for improving the eating quality of cooked rice from a plant of the genus Oryza, comprising at least one method selected from the group consisting of the following (a) to (c): (a) increasing the expression level of mRNA containing a nucleotide sequence having 90% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 1 in rice of the genus Oryza to between 1.3 and 6.0 times the expression level of mRNA containing the nucleotide sequence shown in SEQ ID NO: 1 in Nipponbare rice; (b) increasing the expression level of a protein containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 2 in rice of the genus Oryza to between 1.3 and 6.0 times the expression level of a protein containing the amino acid sequence set forth in SEQ ID NO: 2 in Nipponbare rice; (c) In the genomic DNA of a plant of the genus Oryza, the base corresponding to thymine at position 1463 in the region of the genomic DNA of Nipponbare, whose base sequence is set forth in SEQ ID NO: 3, is replaced with cytosine. [6] The method according to any one of [1] to [5], wherein the rice plant is a progeny rice plant derived from Koshihikari. [7] A primer set for predicting the eating quality of cooked rice from plants of the genus Oryza, comprising a forward primer and a reverse primer amplifying the following regions (A') and / or (C'): (A') a region in the mRNA of a plant of the genus Oryza that contains a part or the entire region whose nucleotide sequence is set forth in SEQ ID NO: 1; (C') A region in the genomic DNA of a rice plant that contains the base corresponding to the 1463rd thymine in the region whose base sequence is set forth in SEQ ID NO: 3 in the genomic DNA of Nipponbare. [8] A plant of the genus Oryza that satisfies the following (a') and / or (b'): (a') the expression level of mRNA in rice containing a nucleotide sequence that has 90% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 1 is more than 2.0 times and not more than 6.0 times the expression level of mRNA containing the nucleotide sequence shown in SEQ ID NO: 1 in Nipponbare rice; (b') The expression level of a protein in rice containing an amino acid sequence that has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2 is more than 2.0 times and not more than 6.0 times the expression level of a protein containing the amino acid sequence shown in SEQ ID NO: 2 in Nipponbare rice. [9] The rice plant according to [8], which is a progeny rice plant derived from Koshihikari.

[10] A rice plant produced by the method described in [4]. [Effects of the Invention]

[0009] According to the present disclosure, there are provided a method for predicting the eating taste of cooked rice from plants of the genus Oryza and a primer set for predicting the eating taste of cooked rice from plants of the genus Oryza. The method for predicting the eating taste of cooked rice from plants of the genus Oryza and the primer set for predicting the eating taste of cooked rice from plants of the genus Oryza according to the present invention make it possible to predict the eating taste of cooked rice from gene expression levels, protein expression levels, or gene sequence. Thus, in producing Oryza plants with excellent cooked rice taste through breeding, even if it is not possible to wait for a cultivation period of about six months to obtain enough rice to evaluate cooked rice taste, it is possible to predict the eating taste of cooked rice from the gene expression levels, protein expression levels, or gene sequence in rice prepared from the seeds or seedlings, and select lines with excellent cooked rice taste.

[0010] The present disclosure provides a method for producing a rice plant with excellent cooked rice taste, and a method for improving the cooked rice taste of a rice plant. The method according to the present invention makes it possible to produce a rice plant with excellent cooked rice taste. This makes it possible to target rice plants with excellent cooked rice taste, for example, in breeding. Furthermore, the method according to the present invention makes it possible to produce a rice plant with excellent cooked rice taste and improve the cooked rice taste, even when rice plants with poor cooked rice taste, such as varieties derived from indica rice varieties native to overseas, are used as parents for breeding. [Brief explanation of the drawings]

[0011] [Figure 1]FIG. 1 shows the relationship between the genotyping results of the 13 cross-breeding progeny plants produced in Example 1, and the evaluation results of the cooked rice taste by sensory testing and the cooked rice taste evaluation results using a cooked rice taste meter. [Figure 2] FIG. 2 shows, in Example 2, the annotation results on RAP-DB for the 75 kbp region narrowed down in Example 1, and the location of SNPs in that region between the genomic DNA of Nipponbare and the genomic DNA of Koshihikari. [Figure 3] This figure shows an excerpt from the results of PLACE analysis of the base sequences of a 3.7 kbp region corresponding to the promoter region upstream of the candidate gene extracted from the total genomic DNA of Nipponbare and Koshihikari. [Figure 4] (A) is a diagram showing the genomic DNA sites targeted by the targeting sequences of the gRNA used in Example 3. (B) is a diagram showing the results of an evaluation of the cooked rice taste of Koshihikari and its three Os03g0108300 knockout lines (T2) in Example 3. [Figure 5] FIG. 10 shows the amount of free xylose in a buffer containing a recombinant protein encoded by the CDS of the Os03g0108300 gene 1, 2, 3, 4, 5, or 10 days after the addition of xyloglucan in Example 4. [Figure 6] FIG. 10 shows the results of analyzing the expression level of Os03g0108300-02 in seeds at the germinating stage using RiceXPro in Example 5. [Figure 7] FIG. 10 shows the results of quantifying Os03g0108300-02 in endosperm-derived mRNA samples 1, 2, 4, and 6 weeks after flowering for a near isogenic line (NIL) in which 495,424 bp of Nipponbare was substituted with that of Koshihikari in Example 5. [Figure 8]6A shows the expression levels of Os03g0108300-02 in wild-type Koshihikari and Aichi Asahi, and in plants overexpressing the CDS sequence of the gene with the RAP-DB ID Os03g0108300, in Example 6. FIG. 6B shows the taste values ​​measured with a rice cooker taste meter in wild-type Koshihikari and Aichi Asahi, and in plants overexpressing the CDS sequence of the gene with the RAP-DB ID Os03g0108300, in Example 6. [Figure 9] (A) is a graph showing the eating quality values ​​of genome-complemented transformants and wild-type Nipponbare in 2018. (B) is a graph showing the eating quality values ​​of genome-complemented transformants and wild-type Nipponbare in 2020. DETAILED DESCRIPTION OF THE INVENTION

[0012] Hereinafter, an embodiment for carrying out the present invention will be described, but the present invention is not limited to the following embodiment.

[0013] In the present disclosure, when a protein or nucleic acid comprises an amino acid sequence or a nucleotide sequence that has 90% or more sequence identity with a given amino acid sequence or a nucleotide sequence, the protein or nucleic acid may have 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity with the given sequence, or in a preferred embodiment, 95% or more sequence identity, and in a most preferred embodiment, 100% sequence identity.

[0014] In the present disclosure, when a sequence contained in a certain protein or nucleic acid has a mutation (i.e., sequence identity is not 100%) with respect to a predetermined amino acid sequence or nucleotide sequence, the mutation may be a mutation selected from substitution, deletion, insertion, and addition in each of 1 to 30 consecutive or dispersed residues or 1 to 100 bases. In a preferred embodiment, the mutation may be a mutation selected from substitution, deletion, insertion, and addition in each of 1 to 10 consecutive or dispersed residues or 1 to 30 bases. In a more preferred embodiment, the mutation may be a mutation selected from substitution, deletion, insertion, and addition in each of 1 to 3 residues or 1 to 10 bases. In an even more preferred embodiment, the mutation may be a mutation selected from substitution, deletion, insertion, and addition in each of 1 to 3 residues or 1 to 10 bases.

[0015] A first embodiment of the present invention relates to a method for predicting the eating quality of cooked rice of a plant of the genus Oryza (hereinafter also referred to as the "prediction method of the first embodiment").

[0016] In this disclosure, unless otherwise limited, "plant" includes whole plants, plant cells, plant protoplasts, plant callus, or plant parts such as embryos, pollen, ovules, gametes, seeds, leaves, flowers, branches, fruits, stems, roots, anthers, etc.

[0017] In the present disclosure, "rice plants" refers to plants belonging to the genus Oryza in the family Poaceae of the order Poales. Representative plants belonging to the genus Oryza include Oryza sativa and Oryza glaberrima, which are widely cultivated for food. The Oryza plant according to the present invention may be, for example, Oryza sativa or Oryza glaberrima, and is preferably Oryza sativa. Oryza sativa may be any of the japonica species (O. sativa subsp. japonica), the indica species (O. sativa subsp. indica), or the javanica species (O. sativa subsp. javanica), and may be, for example, the japonica species.

[0018] The Oryza plant of the present invention may be a hybrid progeny Oryza plant derived from Koshihikari. The Oryza plant of the present invention may be an Oryza plant having Koshihikari or a hybrid progeny Oryza plant derived from Koshihikari as one or both parents. The hybrid progeny Oryza plant derived from Koshihikari may be a first generation hybrid (F1) obtained by crossing Koshihikari as one or both parents, or a second generation hybrid (F2) or later plants obtained by further crossing, for example, F3, F4, F5, F6, F7, F8, F9, F10, F11, F12, F13, F14, F15, F16, F17, F18, F19, F20, F21, F22, F23, F24, F25, F26, F27, F28, F29, F29, F30, F31, F32, F33, F34, F35, F36, F37, F38, F39, F40, F41, F42, F43, F44, F45, F46, F47, F48, F49, F50, F51, F52, F53, F54, F55, F56, F57, F58, F59, F60, F61, F62, F63, F64, F65, F66, F67, F68, F69, F69, F70, F71, F72, F73, F74, F75, F76, F77, F78, F79, F80, F81, F82, F83, F84, F85, F86, F87, F88, F89, F90, F91, F92, F93, F94, F95, F96, F97, 10 For example, it may be a first generation (BC1) plant obtained by backcrossing these hybrids with either of the parent Oryza plants, or a second generation (BC2) or later plant obtained by further backcrossing. Progeny Oryza plants derived from Koshihikari may be F1, F2, F3, F4, or F5 of Koshihikari, and more preferably F1, F2, or F3 of Koshihikari. Known F1 generation varieties of Koshihikari include Hitomebore, Akitakomachi, Hinohikari, and Yumehikari. Known F2 generation varieties of Koshihikari include Kinuhikari, Haenuki, Nanatsuboshi, Masshigura, Yumepirika, and Shimahikari. Known F3 generation varieties of Koshihikari include Kirara 397, Kinumusume, and Moeminori. Therefore, a progeny Oryza plant derived from Koshihikari may be an Oryza plant obtained by crossbreeding Koshihikari or the above-mentioned progeny Oryza plant derived from Koshihikari as one or both parents. Note that "a progeny Oryza plant derived from Koshihikari" does not have to include Koshihikari.

[0019] In this disclosure, "rice" from plants of the genus Oryza refers to rice seeds from which at least the husk has been removed. Rice from plants of the genus Oryza is consumed as a staple food by humans in many countries in Asia, including Japan. The rice according to the present invention is not particularly limited as long as it can be prepared by removing unused parts, including the husk, from seeds of plants of the genus Oryza, contains endosperm, and is edible by humans after cooking. In this disclosure, "rice" from plants of the genus Oryza includes brown rice from which the husk has been removed by hulling or the like. In this disclosure, "rice" from plants of the genus Oryza includes brown rice from which the pericarp, seed coat, and / or aleurone layer (these three are also collectively referred to as "bran layers") have been partially removed by polishing or the like (polished rice), and also includes rice from which all of the pericarp, seed coat, and / or aleurone layer (these three are also collectively referred to as "bran layers") have been removed (germinated rice). In the present disclosure, "rice" of Oryza plants includes brown rice from which the bran layer and germ have been removed (white rice). White rice can also be referred to as polished rice or polished white rice. In addition to these, rice whose cooked rice taste can be predicted by the prediction method of the first embodiment also includes germinated brown rice, for example, brown rice from which sprouts measuring 5 mm or less have sprouted. The rice whose cooked rice taste can be predicted by the prediction method of the first embodiment may be, for example, white rice, germinated rice, polished rice, brown rice, or germinated brown rice, preferably white rice or germinated rice, and more preferably white rice.

[0020] In this disclosure, "cooking rice" refers to cooking rice from plants of the genus Oryza. When humans ingest rice from plants of the genus Oryza, it is common for the rice to be cooked, rather than simply having unnecessary parts removed. Cooking rice is typically performed by soaking the rice in water and heating it at a temperature near 100°C, the boiling point of water, until most of the liquid water used for soaking has evaporated and been absorbed. Cooked rice absorbs water and becomes swollen, with a good texture, and the starch contained in the endosperm gelates, making it easier to absorb as nutrients. Rice cooking according to the present invention may involve heating water and soaked rice, or may involve heating them in the presence of seasonings and / or other ingredients, such as edible parts of animals or plants (e.g., meat, vegetables, seafood, grains, and beans) or processed products thereof. The water used for cooking rice according to the present invention may contain components (e.g., minerals, oxygen, carbonate ions, chlorine) that may be contained in water used for cooking, such as tap water and / or natural water. The water used for cooking rice according to the present invention may be soft water or hard water, or may be soft water.

[0021] In the present disclosure, "cooked rice taste" refers to the taste of rice from a plant of the genus Oryza after cooking. The cooked rice taste according to the present invention may be the taste perceived through the five senses, including taste, when a person eats cooked rice, or may be the taste evaluated using a cooked rice taste meter. The rice from a plant of the genus Oryza according to the present invention may have excellent taste when cooked while soaked in water, or may have excellent taste when cooked while soaked in water and in the presence of seasonings and / or other ingredients, such as edible parts of animals or plants (e.g., meat, vegetables, seafood, grains, and beans) or processed products thereof.

[0022] Taste, as a sensation felt by humans through the five senses including taste when eating rice, may be evaluated according to parameters related to the impression, including taste, that rice gives to humans, for example, by the method described in "Rice Breeding Manual" (co-edited by Yamamoto Ryuichi, Horisuue Noboru, and Ikeda Ryoichi, National Agriculture Research Center Research Materials, No. 30, Yokendo, published in 1996). As an example of a more detailed evaluation, the evaluation may be based on at least one parameter selected from the group consisting of gloss, umami, stickiness, and hardness, which are related to the impression, including taste, that rice gives to humans, or may be based on all of these. In the prediction method of the first embodiment, for example, when an evaluation based on sensations felt by humans through the five senses including taste when eating rice is quantified using the Visual Analogue Scale (VAS), it may be possible to distinguish between two groups with significantly different scores.

[0023] The cooked rice taste meter is not particularly limited as long as it is an instrument capable of evaluating the taste of cooked rice. The cooked rice taste evaluated by the cooked rice taste meter may be, for example, the cooked rice taste measured using a cooked rice taste meter (STA1A, STA1B, etc.) manufactured by Satake Corporation. The cooked rice taste meter manufactured by Satake Corporation is an instrument that can estimate the taste of cooked rice and quantify it as a taste value based on the amount of transmitted light and the amount of reflected light at three wavelengths (Takashi Mikami, "Development of a Rice Taste Quality Evaluation Device," Journal of the Japanese Society of Food Technology, Vol. 10, No. 4, pp. 191-197, 2009). The prediction method of the first embodiment may be capable of distinguishing between two groups whose taste values ​​evaluated by the cooked rice taste meter are significantly different. The prediction method of the first embodiment may be capable of distinguishing between two groups whose average taste values ​​evaluated by the cooked rice taste meter differ by, for example, 3 or more, 5 or more, 10 or more, 15 or more, or 20 or more.

[0024] The prediction method of the first embodiment includes evaluating at least one selected from the group consisting of the following (A) to (C): (A) Expression level of mRNA containing the nucleotide sequence shown in SEQ ID NO: 1 in rice, a plant of the genus Oryza; (B) Expression level of a protein containing the amino acid sequence shown in SEQ ID NO: 2 in rice of the genus Oryza; (C) The base corresponding to the 1463rd thymine in the region of the genomic DNA of the Nipponbare variety whose base sequence is shown in SEQ ID NO: 3 in the genomic DNA of the Oryza plant.

[0025] The mRNA having the nucleotide sequence shown in SEQ ID NO: 1 is identified by the ID number Os03g0108300-02 in the Rice Annotation Project database (RAP-DB), a database for a project aimed at annotating rice genomic DNA. It is the coding sequence (CDS) of a pre-mRNA shown in SEQ ID NO: 4, which is a transcription product of a specific region on the short arm of chromosome 3 of the genomic DNA of plants in the genus Oryza. The protein having the amino acid sequence shown in SEQ ID NO: 2 is a protein obtained by translation of the mRNA shown in SEQ ID NO: 1 in plants in the genus Oryza. The protein having the amino acid sequence shown in SEQ ID NO: 2 has been assigned the gene symbol OsXTH19 and the gene name xyloglucan endotransglucosylase / hydrolase 19 in RAP-DB. The present inventors have confirmed that the protein having the amino acid sequence shown in SEQ ID NO: 2 has enzymatic activity as a xyloglucan degrading enzyme.

[0026] Sequence number 1: ATGGAGCAGAAGCCACCAGCAGTTGCTGCTAATAATAATCAGCTGCTGCTGGATGATGATGATAATGGTGGTGGTGGCGTGCAGTAATTATATGATTAGTGGCGAGGAGCGCAGCCATCGCCGGGATACTACCCGAGCAAGACGATCCGATCGATGGCGTTCGCGGAAGGGTACGACAACCTGTGGGCGGGCAGCCACACACGAGCGCTGCGGCGGACCAGCGCGCTGACGGTTGGATGGATCGGAGCTCCGGCAGCGGCTTCCAAGTCCAAGCGCTCTACCGCAACGGCCTACTTCGGCGCGCTCATCAAGGTCCCCTCCGGCTACACCGGCGTCAACACCGCCTTCTACCTGTCGAACAACGAGCTTACCCGGGCAGCCAGAGATCGACATAGAGCTGCTGGGGACGGTGCGGGGAGCCGTGGACGCTGCAGACGACGAACGTGTACGTGCACGGCA CCGGCGACGGCGCCATCATCGGGAGGGAGATGCGGTTCCACCTCTGGTTCGACCCCACCGCCGACTTCCACCACTACGCCATCCTCTGGAACCCCGACCACATCGTCTTCCTCGTCGACGACGTCCCCGTCCGCCGTTACCCGCGCGCCGCCGGCAACACATTCCCCGACCGCCAGATGGGCCTACGGCTCCATCTGGGACGCCTCCGACTGGGCCACCGACGGCGGCCGCTA CAAGTCCGACTACCGCTACCAGCCCTTCGTCTCGCGCTACCGAGACCTCAAGATCGCGGCTGCGAGGCCGCCGCCGGCGAGCTGCCAGCCCGTGCCGGCGTCGCCGTCGGGCGCCACCGGGCGAGCTCAGCGCGCAGCAGAAGGCGGCCATGAGGTGGGCGCAGCAGAGGTCCATGGTCTACTACTACTGCCAGGACTACTCCAGGAATCACGCAACTACCCCGAGTGCTAG

[0027] SEQ ID NO: 2: MEQKPPAVAANNNQLLLMMIMVVVACSNYMISGAGAQPSPGYYPSKTIRSMAFGEGYDNLWGGQHQTLSADQTALTVWMDRSSGSGFKSKRSYRNGYFGASIKVPSGYTAGVNTAFYLSNNELYPGQHDEIDIELLGTVPGEPWTLQTNVYVHGTGDGAIIGREMRFHLWFDPTADFHHYAILWNPDHIVFLVDDVPVRRYPRAAGNTFPDRQMWAYGSIWDASDWATDGGRYKSDYRYQPFVSRYRDLKIAGCEAAAPASCQPVPASPSGATGELSAQQKAAMRWAQQRSMVYYYCQDYSRNHANYPEC

[0028] The (A) mRNA comprising the nucleotide sequence set forth in SEQ ID NO: 1 in rice of the genus Oryza and the (B) protein comprising the amino acid sequence set forth in SEQ ID NO: 2 in rice of the genus Oryza, the expression levels of which are evaluated in the prediction method of the first embodiment, may contain natural or artificial mutations to the extent that the protein encoded by the mRNA or the xyloglucan degrading activity of the protein is not lost. When these evaluation targets contain mutations, the protein encoded by the mRNA or the xyloglucan degrading activity of the protein may be 0.1-fold or more, 0.3-fold or more, 0.5-fold or more, or 1.0-fold or more, in terms of kcat / Km, of the xyloglucan degrading activity of the protein encoded by the mRNA of the nucleotide sequence set forth in SEQ ID NO: 1 in wild-type rice of Oryza or the protein of the amino acid sequence set forth in SEQ ID NO: 2 in wild-type rice of Oryza. Furthermore, in the prediction method of the first embodiment, (A) the expression level of mRNA containing the base sequence shown in SEQ ID NO: 1 in rice, a plant of the genus Oryza, may be evaluated by quantifying RNA (pre-mRNA) that gives the mRNA after splicing in cells of the plant of the genus Oryza.

[0029] In a preferred aspect, the prediction method of the first embodiment may include measuring the expression level of mRNA in rice, a plant of the genus Oryza, having the base sequence shown in SEQ ID NO: 1. In a preferred aspect, the prediction method of the first embodiment may include measuring the expression level of protein in rice, a plant of the genus Oryza, having the amino acid sequence shown in SEQ ID NO: 2.

[0030] In one embodiment, the expression level of (B) the mRNA of the base sequence set forth in SEQ ID NO: 1 in rice of the genus Oryza and / or the expression level of (B) the protein comprising the amino acid sequence set forth in SEQ ID NO: 2 in rice of the genus Oryza may be the expression level in the embryo or endosperm or seedling of a seed of a plant of the genus Oryza, or may be the expression level in the endosperm of a seed of a plant of the genus Oryza. When the expression level is the expression level in the seed or endosperm, the expression level may be measured in seeds of a plant of the genus Oryza, for example, within 10 weeks from the flowering date. In a preferred embodiment, the expression level may be measured in seeds of a plant of the genus Oryza, for example, within 6 weeks from the flowering date. For example, the expression level may be measured in seeds of a plant of the genus Oryza, for example, within 1, 2, 3, 4, 5, or 6 weeks from the flowering date. For example, the expression level may be measured in seeds of a plant of the genus Oryza, for example, 2 weeks from the flowering date, when the expression level is likely to be highest.

[0031] In the prediction method of the first embodiment, the method for obtaining an RNA-containing sample used to evaluate the expression level of (A) an mRNA containing the base sequence set forth in SEQ ID NO: 1 in rice of the genus Oryza is not particularly limited as long as it is a method capable of extracting RNA from rice of the genus Oryza, and any method commonly used by those skilled in the art can be used. For example, RNA can be extracted from rice of the genus Oryza by the method (CTAB method) described in Murray MG, Thompson WF, Nucleic Acids Research, 8, 4321-4326 (1980). For example, RNA can be extracted from rice of the genus Oryza by disrupting the cell walls of rice cells and then isolating RNA from the resulting cell lysate. Specific methods for extracting RNA from rice of the genus Oryza include, for example, the formic acid phenol extraction method, the sodium dodecyl sulfate phenol method, and the guanidinium thiocyanate method. Commercially available reagents that can be used for this method include TRI Reagent (registered trademark) (Merck) and TRIzol (registered trademark) Reagent (Invitrogen), and RNA can also be extracted from rice, a plant of the genus Oryza, according to the methods described in the instructions for these reagents.

[0032] In the prediction method of the first embodiment, (A) in assessing the expression level of an mRNA containing the nucleotide sequence set forth in SEQ ID NO: 1 in rice, a plant of the genus Oryza, the method for quantifying the mRNA is not particularly limited, as long as it can quantify the mRNA. The mRNA quantification method may be, for example, a PCR method or a method using a next-generation sequencer (NGS). For example, in the PCR method, mRNA can be quantified using the presence and rate of nucleic acid amplification as an indicator by quantitative PCR (e.g., real-time PCR) using a primer set that amplifies an mRNA containing the nucleotide sequence set forth in SEQ ID NO: 1. Alternatively, mRNA can be quantified using fluorescence intensity as an indicator by TaqMan probe method using a similar primer set. Such a primer set is not particularly limited, as long as it can amplify part or all of the nucleotide sequence set forth in SEQ ID NO: 1 and can quantify an mRNA containing the nucleotide sequence set forth in SEQ ID NO: 1. For example, the primer set shown in SEQ ID NO: 5 and SEQ ID NO: 6 can be used. SEQ ID NO: 5: GCGTCAACACCGCCTTCTA (forward primer) SEQ ID NO: 6: CGTCCCCAGCAGCTCTATGT (reverse primer)

[0033] In the prediction method of the first embodiment, the method for obtaining a protein-containing sample used in (B) evaluating the expression level of a protein comprising the amino acid sequence set forth in SEQ ID NO: 2 in rice of the genus Oryza is not particularly limited as long as it is a method that can extract protein from rice of the genus Oryza, and any method commonly used by those skilled in the art can be used. Examples of such protein extraction methods include adding rice or crushed rice to a buffer containing a surfactant, or treating rice or crushed rice with a bead mill or ultrasonic crusher to liberate the protein outside the plant cells, and then isolating the protein by liquid separation using phenol as an organic layer or by protein precipitation by salting out, etc.

[0034] In the prediction method of the first embodiment, the method for quantifying the protein (B) used to evaluate the expression level of the protein comprising the amino acid sequence set forth in SEQ ID NO: 2 in rice of the genus Oryza is not particularly limited as long as it can quantify the protein. The protein quantification method may be, for example, ELISA or Western blotting.

[0035] The present inventors have found that a Nipponbare-derived line produced by transformation in which the expression level of (A) mRNA comprising the nucleotide sequence set forth in SEQ ID NO: 1 in rice of the genus Oryza and / or the expression level of (B) protein comprising the amino acid sequence set forth in SEQ ID NO: 2 in rice of the genus Oryza are increased has better cooked rice taste than Nipponbare. Furthermore, the present inventors have found that in Koshihikari, which has a higher expression level of (A) mRNA comprising the nucleotide sequence set forth in SEQ ID NO: 1 in rice of the genus Oryza and / or (B) protein comprising the amino acid sequence set forth in SEQ ID NO: 2 in rice of the genus Oryza than Nipponbare, further increasing the expression levels of these genes actually deteriorates the cooked rice taste. In one aspect, the prediction method of the first embodiment may predict that a rice plant will have excellent cooked rice taste if the expression level of (A) an mRNA comprising the nucleotide sequence set forth in SEQ ID NO: 1 in rice of a rice plant and / or (B) a protein comprising the amino acid sequence set forth in SEQ ID NO: 2 in rice of a rice plant is within a predetermined multiplier range of the expression level of an mRNA comprising the nucleotide sequence set forth in SEQ ID NO: 1 and / or a protein comprising the amino acid sequence set forth in SEQ ID NO: 2 in rice of a rice plant. The predetermined multiplier range may be, for example, 1.1-fold or more, 1.2-fold or more, 1.3-fold or more, 1.5-fold or more, 1.8-fold or more, 2.0-fold or more, more than 2.0-fold, 2.5-fold or more, or 3.0-fold or more, or may be 7.5-fold or less, 7.0-fold or less, 6.5-fold or less, 6.0-fold or less, 5.5-fold or less, 5.0-fold or less, 4.5-fold or less, or 4.0-fold or less, and these may be freely combined.

[0036] The nucleotide sequence shown in SEQ ID NO: 3 is a promoter region on the short arm of chromosome 3 of the genomic DNA of Nipponbare. The present inventors have discovered that a single nucleotide polymorphism (SNP) exists between Koshihikari and Nipponbare at the base corresponding to thymine at position 1643 of the nucleotide sequence shown in SEQ ID NO: 3, and that when the allele is Koshihikari-type (cytosine), the cooked rice taste is superior to when the allele is Nipponbare-type (thymine). Furthermore, the present inventors have discovered that this SNP improves the cooked rice taste through changes in (A) the expression level of mRNA containing the nucleotide sequence shown in SEQ ID NO: 1 in rice plants of the genus Oryza and / or (B) the expression level of protein containing the amino acid sequence shown in SEQ ID NO: 2 in rice plants of the genus Oryza. That is, in one aspect of the prediction method of the first embodiment, (C) the taste of cooked rice from a rice plant can be predicted by evaluating a polymorphism of a base corresponding to thymine at position 1463 in the region of the genomic DNA of a rice plant whose base sequence is set forth in SEQ ID NO: 3 in the genomic DNA of the Nipponbare variety. For example, if the evaluated base corresponding to thymine at position 1463 is cytosine, it can be predicted that the rice plant will have excellent cooked rice taste. In the base sequence set forth in SEQ ID NO: 3 below, the thymine at position 1643 is indicated in square brackets.

[0037]

[0038] In the prediction method of the first embodiment, (C) the method for obtaining a sample containing genomic DNA used to evaluate the base corresponding to thymine at position 1463 in the region of the genomic DNA of Nipponbare, the base sequence of which is set forth in SEQ ID NO: 3, in the genomic DNA of a plant of the genus Oryza, is not particularly limited as long as it is a method that can extract DNA from an organ of a plant of the genus Oryza. The organ used for sample extraction is not particularly limited and may be, for example, a leaf, stem, seed, or root, or may be a leaf. The sample extraction method is the same as that described for (A) the method for extracting a sample containing RNA used to evaluate the expression level of mRNA containing the base sequence set forth in SEQ ID NO: 1 in rice, a plant of the genus Oryza.

[0039] In the prediction method of the first embodiment, (C) in evaluating the base corresponding to thymine at position 1463 in the region of the genomic DNA of a plant of the genus Oryza, the base sequence of which is set forth in SEQ ID NO: 3 in the genomic DNA of Nipponbare, the method for evaluating the polymorphism (single nucleotide polymorphism) at position 1463 is not particularly limited as long as it is capable of evaluating the polymorphism. Single nucleotide polymorphisms can be detected, for example, by fragment analysis, allele-specific PCR, DNA sequencing, digital PCR, or the like. In fragment analysis, for example, PCR is used to amplify a region of the genomic DNA of a plant of the genus Oryza, containing the base corresponding to thymine at position 1463 in the region of the genomic DNA of Nipponbare, the base sequence of which is set forth in SEQ ID NO: 3. The amplified DNA fragments are then digested with a restriction enzyme, and the resulting cleavage patterns are evaluated by gel electrophoresis or the like to detect SNPs (PCR-RFLP method). For example, allele-specific PCR does not amplify a region containing the base corresponding to thymine at position 1463 in the region of the nucleotide sequence set forth in SEQ ID NO: 3 in the genomic DNA of Nipponbare, but if thymine at position 1463 is substituted with cytosine, PCR using a primer set that amplifies the region can be used to evaluate SNPs using the presence or absence of amplification as an indicator. Furthermore, DNA sequencing can detect SNPs by analyzing the DNA sequence at the SNP site or the sequence of its transcription product using, for example, a next-generation sequencer.

[0040] As mentioned above in the prediction method of the first embodiment, the second embodiment of the present invention is a primer set for predicting the eating quality of cooked rice of plants of the genus Oryza, which is a primer set including a forward primer and a reverse primer that amplify the following regions (A'), (C') and / or (C'') (hereinafter also referred to as the "primer set of the second embodiment"): (A') a region in the mRNA of a plant of the genus Oryza that contains a part or the entire region whose nucleotide sequence is set forth in SEQ ID NO: 1; (C') a region in the genomic DNA of a plant of the genus Oryza that contains a base corresponding to thymine at position 1463 of the region in the genomic DNA of Nipponbare, the base sequence of which is set forth in SEQ ID NO: 3; (C'') A region in the genomic DNA of a rice plant that includes the 1463rd base of the region whose base sequence in the genomic DNA of Nipponbare is shown in SEQ ID NO: 3 (however, it does not amplify if the 1463rd base is thymine, but it amplifies if the 1463rd base is cytosine; i.e., it is an SSR marker (Simple Sequence Repeat marker)).

[0041] In a preferred embodiment, the primer set may include a forward primer and a reverse primer that amplify the following regions (A') and / or (C'): (A') a region in the mRNA of a plant of the genus Oryza that contains a part or the entire region whose nucleotide sequence is set forth in SEQ ID NO: 1; (C') A region in the genomic DNA of a rice plant that contains the base corresponding to the 1463rd thymine in the region whose base sequence is set forth in SEQ ID NO: 3 in the genomic DNA of Nipponbare.

[0042] The base length of the region amplified by these primer sets may be, for example, 10 or more, 30 or more, 50 or more, or 100 or more, or 1000 or less, 500 or less, 300 or less, or 100 or less, and these may be freely combined. In these cases, (A') a primer set comprising a forward primer and a reverse primer that amplifies a region in the mRNA of a plant of the genus Oryza, the region containing part or all of the region whose base sequence is set forth in SEQ ID NO: 1, may be, for example, a primer set consisting of a primer having 90% or more sequence identity to the base sequence set forth in SEQ ID NO: 5 and a primer having 90% or more sequence identity to the base sequence set forth in SEQ ID NO: 6. In these cases, (C') a primer set comprising a forward primer and a reverse primer that amplifies a region in the genomic DNA of a plant of the genus Oryza, the region containing the base corresponding to thymine at position 1463 in the region whose base sequence is set forth in SEQ ID NO: 3 in the genomic DNA of Nipponbare, can be used, for example, in a PCR-RFLP method, together with a restriction enzyme that recognizes only either thymine or cytosine when the base corresponding to thymine at position 1463 is the same as thymine.

[0043] Each primer may have a label detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Detectable labels include, for example, biotin for detection with labeled avidin (e.g., fluorescently labeled streptavidin), haptens, fluorescent dyes (e.g., fluorescein, Texas Red, and rhodamine), electron-dense reagents, enzymes (e.g., horseradish peroxidase and alkaline phosphatase), and radioisotopes (e.g., 32 P, 3 H, 14 C and 125 I) can be mentioned. For example, the label may be present on the 5'-end side, or may be attached via a linker.

[0044] These primers can be produced by methods commonly used by those skilled in the art, for example, by nucleic acid synthesis in liquid phase or solid phase, or can be obtained by outsourcing synthesis to an appropriate synthesis contractor.

[0045] The prediction method of the first embodiment and the primer set of the second embodiment make it possible to predict the cooked rice taste of rice from gene expression levels, protein expression levels, or gene sequences. Thus, in producing an Oryza plant with excellent cooked rice taste through breeding, even if it is not possible to wait for a cultivation period of about six months to obtain a quantity of rice sufficient for evaluating cooked rice taste, it is possible to predict the cooked rice taste of a hybrid progeny plant from the gene expression levels, protein expression levels, or gene sequences in rice prepared from the seeds or seedlings, and select a line with excellent cooked rice taste.

[0046] A third embodiment of the present invention is a method for producing a plant of the genus Oryza that produces rice with excellent cooked rice taste, the method comprising at least one method selected from the group consisting of the following (a) to (c): (a) increasing the expression level of mRNA containing a nucleotide sequence having 90% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 1 in rice of the genus Oryza to between 1.3 and 6.0 times the expression level of mRNA containing the nucleotide sequence shown in SEQ ID NO: 1 in Nipponbare rice; (b) increasing the expression level of a protein containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 2 in rice of the genus Oryza to between 1.3 and 6.0 times the expression level of a protein containing the amino acid sequence set forth in SEQ ID NO: 2 in Nipponbare rice; (c) In the genomic DNA of a plant of the genus Oryza, the base corresponding to thymine at position 1463 in the region of the genomic DNA of Nipponbare, whose base sequence is set forth in SEQ ID NO: 3, is replaced with cytosine.

[0047] As already mentioned above, a plant of the genus Oryza that satisfies at least one requirement selected from the group consisting of (a) to (c) above has excellent cooked rice taste. That is, in one aspect, the third embodiment of the present invention may be a method for improving the cooked rice taste of a plant of the genus Oryza, which may include at least one requirement selected from the group consisting of (a) to (c) above.

[0048] The rice plant, rice, and cooked rice taste in the third embodiment of the present invention are the same as those described in the prediction method of the first embodiment of the present invention.

[0049] In a third embodiment of the present invention, the expression level of an mRNA comprising a nucleotide sequence having 90% or more sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1 and / or the expression level of a protein comprising an amino acid sequence having 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 2 in rice of the genus Oryza may be a predetermined multiplier relative to Nipponbare. The predetermined multiplier range may be, for example, 1.3-fold or more, 1.5-fold or more, 1.8-fold or more, 2.0-fold or more, more than 2.0-fold, 2.5-fold or more, or 3.0-fold or more, or 6.0-fold or less, 5.5-fold or less, 5.0-fold or less, 4.5-fold or less, or 4.0-fold or less, and these may be freely combined. In a preferred aspect of the third embodiment of the present invention, the expression level of an mRNA comprising the nucleotide sequence set forth in SEQ ID NO: 1 and / or the expression level of a protein comprising the amino acid sequence set forth in SEQ ID NO: 2 in rice of the genus Oryza may be a predetermined multiplier relative to Nipponbare.

[0050] In the method of the third embodiment, the protein encoded by the mRNA whose expression level is 1.3 to 6.0 times that of the mRNA containing the nucleotide sequence set forth in SEQ ID NO: 1 in Nipponbare rice, or the protein whose expression level is 1.3 to 6.0 times that of the protein containing the amino acid sequence set forth in SEQ ID NO: 2 in Nipponbare rice, may contain natural or artificial mutations to the extent that the protein encoded by the mRNA or the xyloglucan degrading activity of the protein is not lost. In such cases, the protein encoded by the mRNA or the xyloglucan degrading activity of the protein may be at least 0.1-fold, 0.3-fold, 0.5-fold, or 1.0-fold the xyloglucan degrading activity, in terms of kcat / Km, of the protein encoded by the mRNA containing the nucleotide sequence set forth in SEQ ID NO: 1 in wild-type rice of the genus Oryza or the protein containing the amino acid sequence set forth in SEQ ID NO: 2 in wild-type rice of the genus Oryza.

[0051] In a third embodiment of the present invention, the expression level of an mRNA comprising a nucleotide sequence with 90% or more sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1 and / or the expression level of a protein comprising an amino acid sequence with 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 2 in rice, a plant of the genus Oryza, can be set at a predetermined multiple relative to Nipponbare, for example, by selecting a line from a progeny plant line that has an expression level with a predetermined multiple relative to Nipponbare, or by introducing a gene into cells of the Oryza or modifying the genes of the cells so that the expression level of an mRNA comprising a nucleotide sequence with 90% or more sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1 in rice is set at a predetermined multiple. In the third embodiment of the present invention, the base corresponding to thymine at position 1463 in the genomic DNA of a plant of the genus Oryza, in the region of the nucleotide sequence set forth in SEQ ID NO: 3, can be replaced with cytosine in the genomic DNA of a plant of the genus Oryza, for example, by selecting a line from a progeny plant line in which the base corresponding to thymine at position 1463 is cytosine, or by modifying the genes of cells of the plant of the genus Oryza so that the base corresponding to thymine at position 1463 is cytosine.

[0052] Selection of a line from a hybrid progeny plant strain whose expression level is a predetermined fold higher than that of Nipponbare or whose base corresponding to the 1463rd thymine is cytosine can be achieved, for example, by producing a large number of seeds or seedlings germinated from those seeds that are candidates for a hybrid progeny Oryza strain according to conventional methods, and then evaluating the expression level of mRNA of the base sequence shown in SEQ ID NO: 1, the expression level of protein of the amino acid sequence shown in SEQ ID NO: 2, and / or the base corresponding to the 1463rd thymine in rice prepared from the seeds or seedlings in a manner similar to that used in the prediction method of the first embodiment, and selecting a line whose expression level is a predetermined fold higher than that of Nipponbare and / or whose base corresponding to the 1463rd thymine is cytosine.

[0053] Introduction of a gene into the cells of a plant of the genus Oryza so that the expression level of mRNA containing a base sequence that has 90% or more sequence identity with the base sequence shown in SEQ ID NO: 1 in rice is increased by a predetermined factor, or so that the base corresponding to the 1463rd thymine is changed to cytosine, can be achieved, for example, by introducing into the cells of a plant of the genus Oryza a vector incorporating DNA that serves as a synthesis template for mRNA or its precursor that contains a base sequence that has 90% or more sequence identity with the base sequence shown in SEQ ID NO: 1, or a base sequence in which the base corresponding to the 1463rd thymine is replaced with cytosine, using the Agrobacterium-mediated method or the particle gun method (biolistic method), followed by selection using antibiotics. Genetic modification of cells of a plant of the genus Oryza so that the expression level of mRNA containing a nucleotide sequence with 90% or more sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1 in rice is at a predetermined fold can be achieved, for example, by introducing into cells of a plant of the genus Oryza a vector that modifies the promoter region upstream of the genomic DNA corresponding to the mRNA of the nucleotide sequence set forth in SEQ ID NO: 1 of the plant of the genus Oryza or a vector that modifies the promoter region to increase the number of cis elements therein, using the Agrobacterium-mediated method or the particle gun method (biolistic method), followed by selection using antibiotics. Plant cells that have been gene-introduced or genetically modified in this manner can be cultured and regenerated in a medium containing a plant hormone such as auxin using standard methods to obtain solid plants.

[0054] The method according to the third embodiment of the present invention can produce rice plants with excellent cooked rice taste and can improve the cooked rice taste of rice plants. This can enable the targeted production of rice plants with excellent cooked rice taste, for example, in breeding. Furthermore, the method according to the present invention can produce rice plants with excellent cooked rice taste and improve the cooked rice taste, even when using rice plants with poor cooked rice taste, such as varieties derived from indica rice varieties native to overseas countries, as parents. Furthermore, the method according to the present invention can alter the physical properties of cooked rice, such as stickiness and hardness, without altering the composition or accumulation of starch, the main component of rice, compared to previously reported taste genes (e.g., hypofunctional alleles of the low-amylose gene GBSSI). Therefore, it may be possible to improve the cooked rice taste of rice plants by a method other than modifying the seed storage components of rice plants.

[0055] A fourth embodiment of the present invention is a rice plant that satisfies the following (a') and / or (b'): (a') the expression level of mRNA in rice containing a nucleotide sequence that has 90% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 1 is more than 2.0 times and not more than 6.0 times the expression level of mRNA containing the nucleotide sequence shown in SEQ ID NO: 1 in Nipponbare rice; (b') The expression level of a protein in rice containing an amino acid sequence that has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2 is more than 2.0 times and not more than 6.0 times the expression level of a protein containing the amino acid sequence shown in SEQ ID NO: 2 in Nipponbare rice.

[0056] The rice plant, rice, and cooked rice taste in the fourth embodiment of the present invention are the same as those described in the third embodiment of the present invention. As already mentioned above, such rice plants are excellent in the cooked rice taste. Furthermore, such rice plants can be produced, for example, by the production method according to one aspect of the third embodiment. [Example]

[0057] The present invention will be described in more detail below using examples, but the present invention is not limited to the following examples.

[0058] In the following examples, known rice plants were used: Nipponbare, Koshihikari, and Aichiasahi; and their progeny plants were the 4758 line progeny, corresponding to the BC4F4, BC4F5, BC4F6, or BC4F7 generation, obtained by backcrossing SL811, a CSSL of Nipponbare, as described below, and the 6566 line progeny, corresponding to the BC4F4, BC4F5, BC4F6, BC4F7, or BC4F8 generation, obtained by backcrossing SL609, a CSSL of Koshihikari, as described below. SL811 is a CSSL in which the candidate region for qOE3 on the short arm of rice chromosome 3 in the genomic DNA of Nipponbare is replaced with an allele from Koshihikari. SL609 is a CSSL in which the candidate region for qOE3 on the short arm of rice chromosome 3 in the genomic DNA of Nipponbare was replaced with an allele from Nipponbare (Kiyosumi Hori et al., "Detection of quantitative trait loci controlling pre-harvest sprouting resistance by using backcrossed populations of japonica rice cultivars", Theoretical and Applied Genetics 120, 1547-1557(2010)).

[0059] In the following examples, rice plants were cultivated annually from 2007 to 2023 in a paddy field (36.03°N, 140.11°E) owned by the National Agriculture and Food Research Organization (NARO) in Tsukuba, Ibaraki Prefecture, Japan. Twenty-four plants per line were cultivated in two rows, with an 18 cm spacing between plants within a row and a 36 cm spacing between rows. Each year, rice seeds were sown in mid-April, and the resulting seedlings were transplanted into paddy fields in mid-May for cultivation. Seeds (rice grains) were then harvested at maturity for each line between September and October. Cultivation was carried out according to the standard operating procedures established by the NARO.

[0060] Example 1: Evaluation of cooked rice taste in a group of substitution lines in the candidate region of qOE3 and narrowing down the QTL region We created progeny plants in which a part of the candidate region of qOE3, the QTL region found in Non-Patent Document 1, was substituted, and the QTL region was narrowed down by evaluating the eating quality of the cooked rice.

[0061] [Creation of hybrid progeny plants] SL811 was backcrossed with Nipponbare, yielding 831 progeny lines in the BC4F4 generation, 288 lines in the BC4F5 generation, 59 lines in the BC4F6 generation, and 3700 lines in the BC4F7 generation (a total of 4758 lines). Six representative lines from these progeny lines were used for genotyping and evaluation of cooked rice taste. SL609 was backcrossed with Koshihikari, yielding 524 progeny lines in the BC4F4 generation, 408 lines in the BC4F5 generation, 3450 lines in the BC4F6 generation, 1728 lines in the BC4F7 generation, and 456 lines in the BC4F8 generation (a total of 6566 lines). Seven representative lines from these progeny lines were used for genotyping and evaluation of cooked rice taste.

[0062] [Genotyping] Total DNA was extracted from the leaves of the 13 hybrid progeny plants by the CTAB method according to the method described in the literature (Kiyosumi Hori et al., "Variation in heading date conceals quantitative trait loci for other traits of importance in breeding selection of rice", Breeding Science 62:223-234 (2012)). All of these DNA fragments were amplified by PCR using 11 SSR markers (SEQ ID NOS: 7 to 28) whose sequences are shown in Table 1 below, targeting candidate regions of qOE3, which were selected based on literature on simple sequence repeat (SSR) markers (McCouch, SR et al., "Development and mapping of 2240 new SSR markers for rice (Oryza sativa L.)," DNA Res. 9: 257-279 (2002)) and rice genome information (International Rice Genome Sequencing Project (IRGSP), "The map-based sequence of the rice genome," Nature 436: 793-800 (2005)). The 11 SSR markers (SEQ ID NOS: 7 to 28) whose sequences are shown in Table 1 below, were used according to the method described in the literature (Kiyosumi Hori et al., "Variation in heading date conceals quantitative trait loci for other traits of importance in breeding selection of rice," Breeding Science 62: 223-234 (2012)). The resulting amplification products were electrophoresed on a 3% agarose gel, and the fragment patterns were used to determine whether each genotype was Nipponbare or Koshihikari.

[0063] [Table 1]

[0064] [Preparation of cooked rice for evaluation of cooked rice taste] 500 g of seeds from the 13 hybrid progeny plants were hulled and polished to approximately 90% mass using standard methods. The resulting 350 g of polished rice was washed five times with water, soaked in water for 30 minutes, and then cooked in a rice cooker with three times the amount of water added. The cooked rice prepared in this manner was evaluated for its cooked rice taste using two methods: sensory evaluation and evaluation using a rice cooker taste meter.

[0065] [Sensory test] The sensory evaluation of cooked rice was conducted according to the method described in "Rice Breeding Manual" (co-edited by Yamamoto Ryuichi, Horisuue Noboru, and Ikeda Ryoichi, Agricultural Research Center Research Materials, No. 30, Yokendo, 1996), with approximately 20 judges trained in scoring each component of taste, using the same method as in Non-Patent Document 1. More specifically, approximately 20 judges evaluated the cooked rice taste of the 13 lines developed using the same methods and criteria as in Non-Patent Document 1 in terms of stickiness (ST), hardness (HA), gloss (GL), and taste (TA), using the cooked rice taste of Koshihikari as a control, and an overall evaluation (OE) was obtained from these results. The evaluation results are expressed as a relative value, with Koshihikari being 0, a maximum being 5, and a minimum being -5, with higher values ​​indicating better overall evaluation.

[0066] [Evaluation using a rice cooker] The taste of cooked rice prepared from the 13 lines was evaluated using a rice taste meter (STA1A) manufactured by Satake Corporation. The rice taste meter is a device that can estimate the taste of cooked rice from the amount of transmitted and reflected light at three wavelengths (Takashi Mikami, "Development of a Rice Taste Quality Evaluation Device," Journal of the Japanese Society of Food Science and Technology, Vol. 10, No. 4, pp. 191-197, 2009). The evaluation results are shown as a relative value to the score of Koshihikari, with Koshihikari being given a score of 100 and the lowest being 0.

[0067] Figure 1 shows the relationship between the genotyping results of the 13 cross-breeding progeny plants, the evaluation results of cooked rice taste by sensory testing, and the evaluation results of cooked rice taste using a rice taste meter. In Figure 1, A indicates that the allele amplified by the SSR marker was of the Nipponbare type. In Figure 1, B indicates that the allele amplified by the SSR marker was of the Koshihikari type. Figure 1 revealed that in the candidate region for qOE3 on the short arm of rice chromosome 3, a QTL that indicates good cooked rice taste in the Koshihikari type allele is located in an approximately 75-kbp region between the amplified region of the SSR markers indicated as SNP-38 and RM4108 in Figure 1.

[0068] <Example 2: Annotation of QTL candidate regions> The 75 kbp region narrowed down in Example 1 was annotated based on known databases, and candidate genes that exhibit good eating quality in the Koshihikari-type alleles were searched for.

[0069] First, based on the annotation data of Nipponbare in the Rice Annotation Project database (RAP-DB), a database for a project aimed at annotating rice genomic DNA, annotation was performed on the 75 kbp region narrowed down in Example 1. As a result, 15 candidate genes were extracted as genes that exhibit good eating quality in Koshihikari-type alleles.

[0070] Next, the 75 kbp region narrowed down in Example 1 was compared with the known whole genomic DNA of Nipponbare and Koshihikari to search for polymorphisms. As a result, six single nucleotide polymorphisms (SNPs) were observed in the 75 kbp region. Figure 2 shows the annotation results on RAP-DB for the 75 kbp region narrowed down in Example 1 and the location of SNPs in that region between the genomic DNA of Nipponbare and the genomic DNA of Koshihikari. As shown in Figure 2, unexpectedly, none of the six SNPs discovered overlapped with the genomic regions of the 15 candidate genes identified by annotation. Therefore, it was revealed that no changes occurred in the amino acid sequences of the proteins encoded by these candidate genes, and that the candidate QTLs improve the taste of cooked rice in Koshihikari-type alleles without accompanying changes in protein structure.

[0071] The present inventors suspected that SNPs in the promoter region of genomic DNA may contribute to the improvement of cooked rice taste in Koshihikari-type alleles without altering the protein structure. Therefore, a 3.7-kbp region corresponding to the promoter region upstream of the identified candidate gene was extracted from the known whole genomic DNA of Nipponbare and Koshihikari, and its nucleotide sequence was analyzed using the Database of Plant Cis-acting Regulatory DNA Elements (PLACE, Kenichi Higo et al., "Plant cis-acting regulatory DNA elements (PLACE) database: 1999, Nucleic Acids Research, 1999, Vol. 27, No. 1: 297-300", address: https: / / www.dna.affrc.go.jp / PLACE / ?action=newplace), an online database for searching for cis-elements. An excerpt from the results is shown in Figure 3. In Figure 3, the underlined and boxed portions indicate sequences corresponding to cis-elements that enhance protein expression in the promoter region upstream of rice α-amylase Amy3D. As shown in Figure 3, in the genomic DNA of Koshihikari, the thymine at position 1463 in the region shown in SEQ ID NO: 3 in the genomic DNA of Nipponbare was replaced with cytosine. As a result, a nucleotide sequence complementary to the "CGACG" sequence, known as a cis-element that enhances protein expression in the promoter region upstream of rice α-amylase Amy3D, was found to appear in the nucleotide sequence from positions 1460 to 1464 in the region shown in SEQ ID NO: 3 in the promoter region upstream of one of the identified candidate genes.As a result, it was revealed that in the genomic DNA of Nipponbare, the base sequence from 1432 to 1438 in the region shown in sequence number 3 in the promoter region upstream of the candidate gene contains a base sequence complementary to the ``TATCCAT'' sequence (i.e., one cis element), which is known as one of the cis elements that enhances protein expression in the promoter region upstream of rice α-amylase Amy3D, whereas in the genomic DNA of Koshihikari, in addition to the ``TATCCAT'' sequence, a ``CGACG'' sequence is also present, revealing the presence of two cis elements that enhance the expression of downstream proteins.

[0072] Based on the annotation data of Nipponbare in RAP-DB, the candidate gene downstream of the promoter region into which the cis-element was inserted was the gene with ID Os03g0108300 in RAP-DB.

[0073] Example 3: Contribution of the Os03g0108300 gene to the taste of cooked rice Two variants, Os03g0108300-01 and Os03g0108300-02, are known as transcription products of the Os03g0108300 found in Example 2. It has been suggested that both proteins encoded by these variants have enzymatic activity as a xyloglucan degrading enzyme. The protein encoded by Os03g0108300-02 in particular has been reported to have enzymatic activity as a xyloglucan degrading enzyme, and is assigned the gene symbol OsXTH19 and the gene name xyloglucan endotransglucosylase / hydrolase 19 in the RAP-DB. Therefore, we investigated whether knocking out the Os03g0108300 gene by genome editing would result in a change in the taste of cooked rice obtained from plants of the genus Oryza.

[0074] The Os03g0108300 gene was knocked out by genome editing using the CRISPR / Cas9 system, similar to the method described previously (Satoru Sukegawa et al., "Genome editing in rice mediated by miniature-size Casnuclease SpCas12f," Front. Genome Ed. Mar 13; 5: 1138843 (2023)). The CRISPR / Cas9 vector used, like the vector described previously, contained a sequence (pU6gRNA) for expressing sgRNA via the transcriptional activity of the U6 promoter, and a sequence for co-expressing the next-generation Cas9 nuclease (Cas9ver.2) and hygromycin B phosphotransferase (ZDgRNA-Cas9ver.2-HPT, designed to allow selection of co-expressing cells with hygromycin B). This vector was obtained by first creating pU6gRNA and then inserting it into ZDgRNA to create a sequence containing sgRNA-Cas9ver.2-HPT. The gRNAs used contained three targeting sequences, each of which is shown in Figure 4(A) as the relationship between the targeting sequence and the target genomic DNA site. In Figure 4(A), "6-," "159-," and "239-" indicate the terminal sequences at the 6th, 159th, and 239th bases of Os03g0108300-01 and Os03g0108300-02. Such plasmids were introduced into Agrobacterium by electroporation in a manner similar to that described in the literature (Seiichi Toki et al., "Early infection of scutellum tissue with Agrobacterium allows high-speed transformation of rice", The Plant Journal 47, 969-976 (2006)), and then introduced into Koshihikari cells by Agrobacterium-mediated transformation, followed by regeneration in the usual manner.Three independent transgenic lines were obtained as homozygotes of T2 derived from the three Os03g0108300 knockout lines (T0). As in Example 1, they were cultivated, cooked rice was prepared, and the cooked rice taste was evaluated using a rice taste meter.

[0075] FIG. 4(B) shows the results of evaluating the cooked rice taste of Koshihikari and its three Os03g0108300 knockout lines (T2). In the figure, Koshi-C indicates wild-type Koshihikari, GE-1 / Koshi(6) indicates a line in which the sequence from base 6 in Os03g0108300-01 and Os03g0108300-02 was used as the gRNA targeting sequence in (A) of Figure 4, GE-2 / Koshi(239) indicates a line in which the sequence from base 239 in Os03g0108300-01 and Os03g0108300-02 was used as the gRNA targeting sequence in (A) of Figure 4, and GE-3 / Koshi(159) indicates a line in which the sequence from base 159 in Os03g0108300-01 and Os03g0108300-02 was used as the gRNA targeting sequence in (A) of Figure 4. In the figure, results are shown as mean ± standard deviation for n = 6. * indicates a p value of less than 0.05 in Student's t-test between the wild-type Koshihikari group and the Os03g0108300 knockout line (T2) compared with the wild-type Koshihikari. Figure 4 (B) shows that all three Os03g0108300 knockout lines (T2) had significantly reduced cooked rice taste compared with the wild-type Koshihikari. Therefore, Os03g0108300 is a QTL that conferred good cooked rice taste in the Koshihikari allele.

[0076] Example 4: Activity of the protein encoded by the Os03g0108300 gene We investigated whether the protein encoded by the Os03g0108300 gene has the enzymatic activity of a xyloglucan-degrading enzyme, as previously suggested.

[0077] The coding sequence (CDS) corresponding to the protein encoded by Os03g0108300-02 was obtained from the Os03g0108300 gene in Koshihikari genomic DNA by digestion with EcoRI / XhoI restriction enzymes and then ligated into the pPICZ-C vector (Invitrogen). The resulting vector was then introduced into Pichia pastoris strain KM71H according to standard methods, and the recombinant protein was expressed. The recombinant protein was isolated and purified from Pichia pastoris strain KM71H according to standard methods, and its xyloglucan degradation activity was evaluated. Xyloglucan degradation activity was evaluated by adding 5 μg of xyloglucan (Megazyme) as a substrate to 200 mM McIlvaine buffer containing 150 μg of recombinant protein derived from Pichia pastoris strain KM71H cultured for 1, 2, 3, 4, 5, or 10 days, and reacting at 30°C for 1 hour. The amount of free xylose calculated based on the absorbance at 340 nm was then used as an indicator.

[0078] Figure 5 shows the amount of free xylose after the addition of xyloglucan cultured for 1, 2, 3, 4, 5, and 10 days in a buffer containing a recombinant protein encoded by the CDS of the Os03g0108300 gene. The results in Figure 5 are shown as the mean ± standard deviation for n = 3. The results in Figure 5 show that the amount of free xylose increased in a time-dependent manner, demonstrating that the protein encoded by the CDS of the Os03g0108300 gene indeed has xyloglucan decomposition activity.

[0079] <Example 5: Analysis of expression levels of Os03g0108300-02 in seeds at the germinating stage> Regarding Os03g0108300, which was previously identified as a QTL that indicates good eating quality in cooked rice in Koshihikari-type alleles, we analyzed the expression level of the mRNA, which is a transcript encoding a protein with xyloglucan degrading activity and has the RAP-DB ID Os03g0108300-02 (hereinafter simply referred to as "Os03g0108300-02"), in germinating seeds.

[0080] First, we analyzed the expression level of Os03g0108300-02 in germinating seeds using RiceXPro, a database of rice gene expression data available online (https: / / ricexpro.dna.affrc.go.jp / ). As shown in Figure 6, Os03g0108300-02 was highly expressed in the leaf sheath and endosperm of the seeds. In Figure 6, the number of days indicates the number of days from flowering.

[0081] Next, Os03g0108300-02 was quantified in endosperm-derived mRNA samples 1, 2, 4, and 6 weeks after flowering for Nipponbare and a near isogenic line (NIL) in which 495,424 bp of Nipponbare had been substituted with Koshihikari. Specifically, 2.5 μg of total RNA samples were extracted from endosperms using the standard sodium dodecyl sulfate phenol method. The extracted total RNA was reverse transcribed with oligo(dT)12-18 primer using SuperScript II reverse transcriptase (Invitrogen). cDNA equivalent to 50 ng of total RNA was used as a template for TaqMan PCR reactions using an ABI PRISM 7900 Sequence Detection System (Applied Biosystems, Foster City, CA), and Os03g0108300-02 was quantified by the TaqMan method. The primers shown in SEQ ID NOs: 5 and 6 were used as PCR primers. Nipponbare and NIL were evaluated for three individuals each, n=3. SEQ ID NO: 5: GCGTCAACACCGCCTTCTA (forward primer) SEQ ID NO: 6: CGTCCCCAGCAGCTCTATGT (reverse primer)

[0082] Figure 7 shows the results of quantifying Os03g0108300-02 in endosperm-derived mRNA samples 1, 2, 4, and 6 weeks after flowering for a near isogenic line (NIL) in which the 495,424 bp of Nipponbare was replaced with a Koshihikari-type variant. The vertical axis in Figure 7 represents the mRNA expression level of Os03g0108300-02, relative to the expression level of the rice ubiquitin gene (reference value: 1.0). The results in Figure 7 show that the NIL in which the 495,424 bp of Nipponbare was replaced with a Koshihikari-type variant had higher levels of Os03g0108300-02 in the endosperm than Nipponbare. These results confirm that the SNP discovered in Example 3 increases the expression level of the protein with xyloglucan degradation activity encoded by Os03g0108300-02.

[0083] Example 6: Changes in taste due to enhanced expression of Os03g0108300-02 The effect of Os03g0108300-02 on the eating quality of cooked rice was further investigated by evaluating the eating quality of rice plants with enhanced expression of Os03g0108300-02.

[0084] First, the entire CDS sequence of the DNA identified by the RAP-DB ID Os03g0108300 was obtained using standard methods, amplified by PCR, and then introduced into the pPZP-Ha3(+) vector (Takuichi Fuse et al., "Ti-Plasmid Vectors Useful for Functional Analysis of Rice Genes," Plant Biotechnology 18(3):219-222 (2001)). The vector thus constructed was then introduced into Koshihikari (Koshi) or Aichi Asahi (Aichi) by Agrobacterium-mediated transformation, as in Example 3. Three overexpression variants (Ox-1 to Ox-3) were prepared for each of Koshihikari and Aichi Asahi. These and the wild-type (VC) were cultivated, cooked, and evaluated for cooked rice taste using a rice taste meter in 2017 and 2019, as in Example 1. Furthermore, for endosperm 2 weeks after flowering, Os03g0108300-02 was quantified in endosperm-derived mRNA samples in the same manner as in Example 5.

[0085] Figure 8 (A) shows the expression levels of Os03g0108300-02 in wild-type Koshihikari and Aichi Asahi, and in plants overexpressing the CDS sequence of the gene with the RAP-DB ID Os03g0108300. Figure 8 (B) shows the taste values ​​of wild-type Koshihikari and Aichi Asahi, and in plants overexpressing the CDS sequence of the gene with the RAP-DB ID Os03g0108300, measured using a rice cooker taste meter. In these figures, results are shown as the mean ± standard deviation for n = 6, and * indicates a p-value of less than 0.05 between the wild-type Koshihikari and the wild-type Aichi Asahi in Student's t-test. According to Figures 8 (A) and (B), unexpectedly, when the expression level of Os03g0108300-02 is within a certain range of multiplication (for example, about 2.0 times) relative to Koshihikari or Aichi Asahi, the taste value is further improved relative to Koshihikari and Aichi Asahi, which are known to have excellent cooked rice taste. On the other hand, when the expression level of Os03g0108300-02 is excessively high (for example, about 4 to 5 times) relative to Koshihikari or Aichi Asahi, the taste value is actually reduced.

[0086] We also investigated whether replacing the promoter region upstream of Os03g0108300 in Nipponbare with the allele from Koshihikari would improve palatability. A 5.7-kbp genomic fragment upstream of the promoter region upstream of Os03g0108300 in Koshihikari was obtained by standard digestion with EcoRI / XhoI restriction enzymes and then ligated into the pPZP2H-lac binary vector (Takuichi Fuse et al., "Ti-Plasmid Vectors Useful for Functional Analysis of Rice Genes," Plant Biotechnology 18(3):219-222 (2001)). The resulting vector was then introduced into Nipponbare (Nichi) by Agrobacterium-mediated transformation, as in Example 3, to obtain genome-complementary transformants. As in Example 1, these, the wild type, and Nipponbare (VC) were cultivated in 2018 (3 transformants and the wild type) and 2020 (7 transformants and the wild type), and cooked rice was prepared, and the cooked rice taste was evaluated using a rice taste meter.

[0087] Figure 9 (A) shows the eating quality values ​​of the genome-complemented transformants and wild-type Nipponbare in 2018. Figure 9 (B) shows the eating quality values ​​of the genome-complemented transformants and wild-type Nipponbare in 2020. In these figures, results are shown as the mean ± standard deviation for n = 6, and * indicates a p-value of less than 0.05 between the transformants and wild-type Nipponbare in a Student's t-test. Figures 9 (A) and (B) show that the genome-complemented transformants in which the promoter region upstream of Os03g0108300 was replaced with a Koshihikari allele had better cooked rice taste than wild-type Nipponbare. This demonstrates that transforming the promoter region upstream of Os03g0108300 to the Koshihikari type improves the eating quality of cooked rice in Oryza sativa plants.

Claims

1. A method for predicting the eating quality of cooked rice from a plant of the genus Oryza, comprising evaluating at least one selected from the group consisting of the following (A) to (C): (A) the expression level of mRNA containing the base sequence shown in SEQ ID NO: 1 in rice of the genus Oryza; (B) the expression level of a protein comprising the amino acid sequence shown in SEQ ID NO: 2 in rice of the genus Oryza; (C) A base corresponding to the 1463rd thymine in the region of the genomic DNA of a rice plant whose base sequence is shown in SEQ ID NO: 3 in the genomic DNA of Nipponbare.

2. the method comprises evaluating (A) the expression level of mRNA comprising the base sequence shown in SEQ ID NO: 1 in rice, a plant of the genus Oryza, and / or (B) the expression level of a protein comprising the amino acid sequence shown in SEQ ID NO: 2 in rice, a plant of the genus Oryza; The method of claim 1, further comprising predicting that the rice plant will have excellent cooked rice taste if the expression level of the mRNA and / or the protein in the evaluated rice of the rice plant is 1.3 to 6.0 times the expression level of the mRNA containing the base sequence shown in SEQ ID NO: 1 and / or the protein containing the amino acid sequence shown in SEQ ID NO: 2 in Nipponbare rice.

3. the method includes (C) evaluating a base in the genomic DNA of a plant of the genus Oryza that corresponds to thymine at position 1463 in the region of the genomic DNA of Nipponbare, the base sequence of which is set forth in SEQ ID NO: 3; The method according to claim 1, further comprising predicting that the rice plant will have excellent cooked rice taste if the evaluated base corresponding to the 1463rd thymine is cytosine.

4. A method for producing a plant of the genus Oryza that produces rice with excellent cooked rice taste, the method comprising at least one method selected from the group consisting of the following (a) to (c): (a) increasing the expression level of mRNA containing a base sequence having 90% or more sequence identity to the base sequence shown in SEQ ID NO: 1 in rice of the genus Oryza to between 1.3 and 6.0 times the expression level of mRNA containing the base sequence shown in SEQ ID NO: 1 in Nipponbare rice; (b) increasing the expression level of a protein comprising an amino acid sequence having 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 2 in rice of the genus Oryza to between 1.3 and 6.0 times the expression level of a protein comprising the amino acid sequence set forth in SEQ ID NO: 2 in Nipponbare rice; (c) In the genomic DNA of a plant of the genus Oryza, the base corresponding to thymine at position 1463 in the region of the genomic DNA of Nipponbare, whose base sequence is set forth in SEQ ID NO: 3, is replaced with cytosine.

5. A method for improving the eating quality of cooked rice from a plant of the genus Oryza, comprising at least one method selected from the group consisting of the following (a) to (c): (a) increasing the expression level of mRNA containing a base sequence having 90% or more sequence identity to the base sequence shown in SEQ ID NO: 1 in rice of the genus Oryza to between 1.3 and 6.0 times the expression level of mRNA containing the base sequence shown in SEQ ID NO: 1 in Nipponbare rice; (b) increasing the expression level of a protein comprising an amino acid sequence having 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 2 in rice of the genus Oryza to between 1.3 and 6.0 times the expression level of a protein comprising the amino acid sequence set forth in SEQ ID NO: 2 in Nipponbare rice; (c) In the genomic DNA of a plant of the genus Oryza, the base corresponding to thymine at position 1463 in the region of the genomic DNA of Nipponbare, whose base sequence is set forth in SEQ ID NO: 3, is replaced with cytosine.

6. The method according to any one of claims 1 to 5, wherein the rice plant is a progeny rice plant derived from Koshihikari.

7. A primer set for predicting the eating quality of cooked rice from plants of the genus Oryza, comprising a forward primer and a reverse primer amplifying the following regions (A') and / or (C'): (A') a region in the mRNA of a plant of the genus Oryza, the region containing a part or the whole of the region whose nucleotide sequence is set forth in SEQ ID NO: 1; (C') A region in the genomic DNA of a plant of the genus Oryza that contains a base corresponding to the 1463rd thymine in the region whose base sequence is set forth in SEQ ID NO: 3 in the genomic DNA of Nipponbare.

8. A rice plant that satisfies the following (a') and / or (b'): (a') the expression level of mRNA in rice comprising a base sequence having 90% or more sequence identity to the base sequence shown in SEQ ID NO: 1 is more than 2.0 times and not more than 6.0 times the expression level of mRNA comprising the base sequence shown in SEQ ID NO: 1 in Nipponbare rice; (b') The expression level of a protein in rice containing an amino acid sequence that has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2 is more than 2.0 times and not more than 6.0 times the expression level of a protein containing the amino acid sequence shown in SEQ ID NO: 2 in Nipponbare rice.

9. The rice plant according to claim 8, which is a progeny rice plant derived from Koshihikari.

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