A novel short-culm gene in grasses that is effective for increasing yields

Abstract

To provide a novel gramineous plant dwarf gene used to create a plant having both falling resistance and productivity.SOLUTION: The present invention provides a method to select a dwarf gramineous plant that determines the existence of d64 gene by detecting a DNA marker present in a region with a distance of 3.3-5.6 Mb from the 5' short arm end of a first chromosome of a gramineous plant.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a novel gene for shortening culms in grasses that is effective in increasing yields. [Background technology]

[0002] In recent years, with the intensifying climate change and frequent typhoons causing rice plants to lodge, reducing yields, has become a problem, and there is a need to improve rice's lodging resistance or robustness. About 60 genes that show short culms are known to improve rice's lodging resistance or robustness, but most of them also have characteristics such as extreme dwarfism or small grains, so they do not lead to increased yields.

[0003] Currently, sd1 and d60 (Patent Document 1) are known to be beneficial short-culm genes, but in light of the breeding philosophy of expanding genetic diversity, it is necessary to search for new sources of short-culm genes in addition to the current limited sources. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 6056042 specification Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide a novel rice short-culm gene that can be used to create plants that have both lodging resistance and productivity. [Means for solving the problem]

[0006] The inventors genetically and genomically analyzed the short-culm gene in "Koganebare," which inherits the short-culm trait from Nipponbare, and "Isehikari," the only mutant variety at Ise Shrine that did not fall over when a typhoon hit. As a result, they discovered a novel short-culm gene, which they named "d64."

[0007] Therefore, the present invention includes the following embodiments. [1] A method for selecting short-culm grasses, characterized by determining the presence of the d64 gene by detecting a DNA marker located in a region 3.3 to 5.6 Mb from the end of the 5' short arm of chromosome 1 of the grasses. [2] The method according to [1], wherein the DNA marker is RM10183. [3] The method according to [1], wherein the DNA marker is a SNP located at a position selected from the group consisting of 3,380,548 base positions, 5,534,571 base positions, and 5,538,047 base positions from the 5' short arm end of chromosome 1 of a grass plant. [4] The method according to [3], wherein the SNP is selected from the group consisting of a T to C substitution at base position 3,380,548, a C to G substitution at base position 5,534,571, and a C to T substitution at base position 5,538,047. [5] A method for producing a short-culm grass plant, comprising: (a) crossing a first parent grass plant having a short culm trait with a second parent grass plant to obtain a hybrid plant; (b) crossing said hybrid plants with each other, or backcrossing or multi-crossing said hybrid plants to obtain progeny plants; (c) further crossing between progeny plants, or backcrossing or multi-line crossing with progeny plants may be performed repeatedly; (d) selecting short-culm grass plants for plants of any or all generations by the method described in any one of [1] to [4]. A method comprising: [6] The method according to [5], wherein the first parent grass plant is a short-culm grass plant containing a mutation at one or more positions selected from the group consisting of 3,380,548 base positions, 5,534,571 base positions, and 5,538,047 base positions from the 5' short arm end of chromosome 1. [7] The method according to [6], wherein the mutation is selected from the group consisting of a T to C substitution at base position 3,380,548, a C to G substitution at base position 5,534,571, and a C to T substitution at base position 5,538,047. [8] The method according to any one of [5] to [7], wherein the second parent grass family plant is a grass family plant that does not have the trait of short culm. [9] A short-culm grass plant having a mutation introduced into a position selected from the group consisting of 3,380,548 bases, 5,534,571 bases, and 5,538,047 bases from the end of the 5' short arm of chromosome 1.

[10] The short-culm grass plant according to [9], wherein the mutation is selected from the group consisting of a T to C substitution at 3,380,548 nucleotides, a C to G substitution at 5,534,571 nucleotides, and a C to T substitution at 5,538,047 nucleotides.

[11] A DNA marker comprising an SNP at a position selected from the group consisting of 3,380,548 base positions, 5,534,571 base positions, and 5,538,047 base positions from the 5' short arm end of chromosome 1 of a grass plant.

[12] The DNA marker according to

[11] , wherein the SNP is selected from the group consisting of a T to C substitution at 3,380,548 bases, a C to G substitution at 5,534,571 bases, and a C to T substitution at 5,538,047 bases.

[13] A probe or primer for detecting the DNA marker according to

[11] or

[12] . [Effects of the Invention]

[0008] According to the present invention, by detecting the presence of the d64 gene using a DNA marker on chromosome 1 of a grass plant, short-culm grass plants can be easily selected. It has been found that such short-culm grass plants have the advantageous characteristics of being shorter than Koshihikari, having grain size equivalent to Koshihikari, and having a greater number of panicles than Koshihikari. Therefore, according to the present invention, grass plants with excellent lodging resistance and high yielding properties can be obtained. [Brief explanation of the drawings]

[0009] [Figure 1] This shows the SNP frequency distribution indicating the relative positions of sd1 and d64 on chromosome 1 in double short-culm Koshihikari (Koshihikari sd1d64), obtained by whole-genome analysis. [Figure 2] A comparison of Koshihikari with d64 introduced (Koshihikari d64), Koshihikari with sd1 introduced, and double short-culm Koshihikari (Koshihikari sd1d64) is shown. [Figure 3] These are photographs of Koshihikari with d64 introduced (Koshihikari d64), Koshihikari with sd1 introduced, double short-culm Koshihikari (Koshihikari sd1d64), and Koshihikari. DETAILED DESCRIPTION OF THE INVENTION

[0010] The novel short-culm gene d64 was discovered using the following method. In the F2 cross between Koshihikari and Koganebare, Koganebare-type short-culm individuals and Koganebare-type long-culm individuals segregated at a 1:3 ratio. Meanwhile, in the F2 cross between Koshihikari sd1 (Hikari Shinseiki), an isogenic line of Koshihikari that contains sd1, and Koganebare, and in the F2 cross between Koshihikari sd1 and Isehikari, a double short-culm type even shorter than Koshihikari sd1 was segregated. This revealed the existence of a short-culm gene distinct from sd1, which was named "d64." Furthermore, since culm length did not segregate in the F2 cross between Isehikari and Koganebare, these varieties were found to share the same short-culm gene d64.

[0011] Next, we selected the Koganebare-type short-culm individuals, the Koganebare-derived double-culm short-culm individuals, and the Isehikari-derived double-culm short-culm individuals from the F2 hybrids. We then backcrossed these individuals six times to Koshihikari and Koshihikari sd1, resulting in the development of "Koshihikari d64-k," "Koshihikari sd1 d64-k," and "Koshihikari sd1 d64-i," in which 99.2% of the genome was replaced by the Koshihikari genome. We also developed an isogenic line, "Koshihikari Hd16," in which only the late-maturing gene Hd16 from the late-maturing, short-culm Isehikari was introgressed. Next, we performed whole-genome analysis of these isogenic lines with the Koshihikari genetic background to search for SNPs (Single Nucleotide Polymorphisms) that were common to "Koshihikari d64-k," "Koshihikari sd1d64-k," and "Koshihikari sd1d64-i" but not present in "Koshihikari Hd16," and that were of Nipponbare origin. As a result, the SNPs that met the above criteria were narrowed down to three locations in the terminal region of the short arm of chromosome 1 and one location on chromosome 7. Furthermore, we examined the linkage of known DNA markers near these candidate SNPs to the Koganebare-type short culm. We found that the SSR marker RM10183 (3.745 Mb) on chromosome 1 was strongly linked to the Koganebare-type short culm. These results indicate that d64 is caused by three SNPs at the end of the short arm of chromosome 1, namely, at 3,380,548 bp, 5,534,571 bp, and 5,538,047 bp from the 5' end of the short arm of chromosome 1. Of these, the 3,380,549 bp SNP is included in the gene region encoding the transcription factor R2R3-MYB, so d64 is predicted to be associated with mutations in the R2R3-MYB gene.

[0012] Therefore, d64 is a short-culm gene located in a region 3.3 to 5.6 Mb from the end of the 5' short arm of chromosome 1 of grasses. As used herein, the term "short-culm gene" refers to a gene that causes short culms in grasses or a gene involved in short culm formation in grasses.

[0013] The d64 gene has a base sequence containing a mutation at one or more base positions selected from the group consisting of 3,380,548 base positions, 5,534,571 base positions, and 5,538,047 base positions from the 5' short arm of chromosome 1. As used herein, "mutation" includes substitution, deletion, and insertion. For example, the mutation may be one or more mutations selected from the group consisting of a substitution of T (thymine) at the 3,380,548 base position with C (cytosine), G (guanine), or A (adenine), a substitution of C at the 5,534,571 base position with G, A, or T, and a substitution of C at the 5,538,047 base position with T, G, or A, compared to the genomic sequence of a grass family plant that does not have d64. Preferably, the mutation may be one or more mutations selected from the group consisting of a T to C substitution at the 3,380,548 base position, a C to G substitution at the 5,534,571 base position, and a C to T substitution at the 5,538,047 base position, compared to the genomic sequence of a grass plant that does not have d64.

[0014] In the present specification, examples of "grass plants not having d64" or "lines not having the d64 gene" include Koshihikari, Hitomebore, Tsuyahime, and Kinuhikari.

[0015] One aspect of the present invention provides a method for selecting short-culm grass plants (hereinafter also referred to as the "method for selecting short-culm grass plants of the present invention"), which is characterized by determining the presence of the d64 gene by detecting a DNA marker present in a region 3.3 to 5.6 Mb from the end of the 5' short arm of chromosome 1 of the grass plant.

[0016] As used herein, "short-culm grasses" refers to grasses with a shortened height, preferably grasses with normal panicle length and grain size but shortened height. "Gramineous plants" also include, but are not limited to, rice, wheat, barley, oat, rye, millet, foxtail millet, barnyard millet, corn, finger millet, sorghum, and the like. Rice includes cultivated and wild species, and preferably cultivated rice, for example, but not limited to, various varieties and strains belonging to Oryza sativa L., can be used.

[0017] "DNA marker" refers to a DNA polymorphism that can be used as a marker for genetic analysis and exhibits differences in nucleotide sequence between individuals or strains, such as RFLP (restriction fragment length polymorphism), AFLP (amplified fragment length polymorphism), SSR (simple sequence repeat), STS (sequence tagged site), SNP (single nucleotide polymorphism), and RAPD (random amplified fragment polymorphism). Known DNA markers can be searched, for example, from public databases.

[0018] In the method for selecting short-culm grasses of the present invention, the "DNA marker located in a region 3.3 to 5.6 Mb from the end of the 5' short arm of chromosome 1" refers to, for example, a DNA marker linked to the d64 gene at a low recombination titer (e.g., 15% or less, preferably 10% or less). This DNA marker can be obtained, for example, by developing an F2 cross between a line containing the d64 gene and a line lacking the d64 gene, identifying DNA markers located in a region 3.3 to 5.6 Mb from the end of the 5' short arm on chromosome 1 that exhibit a specific pattern in the genomic DNA obtained from the d64 homozygous F2 individuals, and determining the recombination titer for the DNA markers that exhibit this specific pattern. Furthermore, DNA markers that exhibit this specific pattern can be backcrossed to determine the genome substitution rate, and those with a low substitution rate, for example, 50% or less, can be used as DNA markers. The recombination titer can be calculated using the following formula: Recombination value (%) = (number of recombinant gametes / total number of gametes) × 100

[0019] For example, RM10183 can be used as the DNA marker. RM10183 is an SSR marker located 3.745 Mb from the end of the 5' short arm of chromosome 1.

[0020] Another example of a "DNA marker located in a region 3.3 to 5.6 Mb from the end of the 5' short arm of chromosome 1" in the method for selecting short-culm grasses of the present invention is a SNP located at one or more bases selected from the group consisting of 3,380,548 bases, 5,534,571 bases, and 5,538,047 bases from the end of the 5' short arm of chromosome 1 of a grass. Further examples of such DNA markers include DNA markers containing one or more SNPs selected from the group consisting of a T to C, G, or A substitution at 3,380,548 bases from the end of the 5' short arm of chromosome 1, a C to G, A, or T substitution at 5,534,571 bases from the end of the 5' short arm of chromosome 1, and a C to T, G, or A substitution at 5,538,047 bases from the end of the 5' short arm of chromosome 1, compared to the genomic sequence of a grasseous plant that does not have d64. Further examples of the DNA markers that can be used include one, two, or three SNPs selected from the group consisting of a T to C substitution at 3,380,548 bases from the 5' end of the short arm of chromosome 1, a C to G substitution at 5,534,571 bases from the 5' end of the short arm of chromosome 1, and a C to T substitution at 5,538,047 bases from the 5' end of the short arm of chromosome 1, compared to the genomic sequence of a grass plant that does not have d64. These SNP markers are novel DNA markers.

[0021] Therefore, in another embodiment, the present invention provides a DNA marker for determining the presence of the d64 gene (hereinafter also referred to as "the DNA marker of the present invention"), which comprises an SNP at a position selected from the group consisting of 3,380,548 bases, 5,534,571 bases, and 5,538,047 bases from the 5' end of Chromosome 1 of a grass plant. As an example of the DNA marker of the present invention, there is provided a DNA marker comprising an SNP selected from the group consisting of a T to C, G, or A substitution at 3,380,548 bases from the 5' end of Chromosome 1, a C to G, A, or T substitution at 5,534,571 bases from the 5' end of Chromosome 1, and a C to T, G, or A substitution at 5,538,047 bases from the 5' end of Chromosome 1, compared to the genomic sequence of a grass plant that does not have d64. Further examples of DNA markers of the present invention include DNA markers containing SNPs selected from the group consisting of a T to C substitution at 3,380,548 bases from the end of the 5' short arm of chromosome 1, a C to G substitution at 5,534,571 bases from the end of the 5' short arm of chromosome 1, and a C to T substitution at 5,538,047 bases from the end of the 5' short arm of chromosome 1, compared to the genomic sequence of a grass plant that does not have d64.

[0022] In the method for selecting short-culm grasses of the present invention, "detection" may be performed using a method commonly used in the art for detecting DNA markers, and those skilled in the art can appropriately select a method depending on the type of DNA marker used. Examples include PCR, Southern blotting, and AFLP.

[0023] In the case of SSR markers, for example, PCR can be performed using primers specific to each SSR and DNA extracted from the sample as a template, and the length of the amplified product can be detected to determine whether the sample contains the target gene.

[0024] In the case of SNP markers, for example, a probe that specifically hybridizes to each SNP allele is designed, and the end of the probe is modified with a different fluorescent dye and quencher for each SNP allele. Detection can be achieved by PCR using a primer designed outside the probe. The probe is degraded by the extension reaction of the primer, and the fluorescence emitted by the separation of the fluorescent dye and quencher is measured, thereby obtaining an allele-specific fluorescence amplification curve and determining which SNP allele the sample has. Examples of fluorescent dyes that can be used include, but are not limited to, FAM and HEX. Allele-specific probes and primers can be appropriately designed by those skilled in the art based on the genomic information of the sample. SNP markers can be detected by known methods, including, but not limited to, TaqMan PCR, Invader PCR, single-base extension, and pyrosequencing.

[0025] Therefore, as another aspect of the present invention, a probe or primer for detecting the DNA marker of the present invention is provided.Such probe and primer can be easily designed and prepared by methods known in the art.For example, but not limited to, a fluorescent dye-attached probe that binds to about 17 bp containing the SNP and a pair of about 20 bp primers that bind about 100 bp before and after the SNP are provided.

[0026] The method for selecting a short-culm grass plant of the present invention is used, for example, in a breeding method for producing a novel short-culm grass plant. Examples of such a breeding method include general methods known in the art. Therefore, one aspect of the present invention is a method for producing a short-culm grass plant, comprising: (a) crossing a first parent grass plant having a short culm trait with a second parent grass plant to obtain a hybrid plant; (b) crossing said hybrid plants with each other, or backcrossing or multi-crossing said hybrid plants to obtain progeny plants; (c) furthermore, the progeny plants may be repeatedly crossed with each other, or backcrossed or multi-line crossed with the progeny plants; (d) Selecting short-culm grass plants for any or all generations of plants by the method for selecting short-culm grass plants of the present invention. The present invention provides a method (hereinafter also referred to as "the method for producing a short-culm grass plant of the present invention") comprising:

[0027] In the method for producing a short-culm grass plant of the present invention, a grass plant having d64 is used as the "first parent grass plant having the trait of short culm." Examples of grass plants having d64 include, but are not limited to, Koganbare, Isehikari, and Nipponbare, as well as short-culm grass plants obtained by selection using the method for selecting short-culm grass plants of the present invention. Furthermore, as a grass plant having d64, a short-culm grass plant containing a mutation at one or more positions selected from the group consisting of 3,380,548 bases, 5,534,571 bases, and 5,538,047 bases from the 5' short arm end of chromosome 1 can be used. The mutation is determined by comparing the genome sequence of a grass plant that does not have d64. For example, but not limited to, a short-culm grass may be used that contains a mutation selected from the group consisting of a T to C, G, or A substitution at 3,380,548 bases from the end of the 5' short arm of chromosome 1, a C to G, A, or T substitution at 5,534,571 bases from the end of the 5' short arm of chromosome 1, and a C to T, G, or A substitution at 5,538,047 bases from the end of the 5' short arm of chromosome 1, compared to the genomic sequence of a grass that does not have d64. For example, a short-culm grass may be used that contains a mutation selected from the group consisting of a T to C substitution at 3,380,548 bases from the end of the 5' short arm of chromosome 1, a C to G substitution at 5,534,571 bases from the end of the 5' short arm of chromosome 1, and a C to T substitution at 5,538,047 bases from the end of the 5' short arm of chromosome 1, compared to the genome sequence of a grass that does not have d64.

[0028] In the method for producing a short-culm grass plant of the present invention, the "second parent grass plant" may be any grass plant and is not particularly limited. For example, a grass plant that does not have the short-culm trait may be used as the second parent grass plant. Examples of grass plants that do not have the short-culm trait include, but are not limited to, Koshihikari, Hitomebore, Tsuyahime, and Kinuhikari. Alternatively, a grass plant that has the short-culm trait may be used as the second parent grass plant. Such short-culm grass plants may include grass plants that have d64, grass plants that have a short-culm gene other than d64, grass plants that have two or more short-culm genes, such as d64 and other short-culm genes, or short-culm grass plants whose cause of the short-culm trait is unknown. Examples of short-culm genes other than d64 include, but are not limited to, sd1 and d60.

[0029] In the method for producing short-culm grasses of the present invention, an F1 hybrid plant is obtained by crossing a first parent plant with a second parent plant, and then the F1 hybrid plants are crossed with each other, or backcrossed or multi-line crossed with the F1 hybrid plant to obtain progeny plants. Further crosses between the progeny plants, or backcrossed or multi-line crosses with the progeny plants, may be performed to obtain further progeny plants. Furthermore, further crosses between the further progeny plants, or backcrossed or multi-line crosses with the further progeny plants may be performed. In this way, crosses between progeny plants, and backcrosses and multi-line crosses with progeny plants may be performed repeatedly; the number of times is not particularly limited and may be determined appropriately by one skilled in the art.

[0030] In backcrossing, one parent plant is a first or second parent grass family plant, or a grass family plant other than the first or second parent grass family plant. In multi-line crossing, one parent plant is a grass family plant other than the first or second parent grass family plant. The "grass family plant other than the first or second parent grass family plant" is not particularly limited as long as it is a grass family plant different from the first or second parent grass family plant, and may be any grass family plant. In backcrossing and multi-line crossing, any number of types of parent plants may be included, and the number of parent plants is not limited.

[0031] For example, a grass plant that does not have the short culm trait may be used as a "grass plant other than the first or second parent grass plant." Examples of grass plants that do not have the short culm trait include, but are not limited to, Koshihikari, Hitomebore, Tsuyahime, and Kinuhikari. Alternatively, a grass plant that has the short culm trait may be used as a "grass plant other than the first or second parent grass plant." Such short culm grass plants may include grass plants with d64, grass plants with a short culm gene other than d64, grass plants with two or more short culm genes, such as d64 and other short culm genes, or short culm grass plants whose cause of the short culm trait is unknown. Examples of short culm genes other than d64 include, but are not limited to, sd1 and d60.

[0032] When backcrossing is performed multiple times, the same parent grass plant may be used as the recurrent parent, or different parent grass plants may be used, and one skilled in the art can make an appropriate selection.

[0033] In the method for producing a short-culm grass plant of the present invention, selection by the method for selecting a short-culm grass plant of the present invention may be performed in any of the generations obtained by the above steps (a), (b), and (c). The selected short-culm grass plant is used in the next crossbreeding. By increasing the number of times of selection by the method for selecting a short-culm grass plant of the present invention, for example by performing the selection in all generations, more accurate selection of short-culm grass plants becomes possible, and short-culm grass plants can be obtained more efficiently.

[0034] Furthermore, one aspect of the present invention provides a novel short-culm grass plant into which the d64 gene has been introduced (hereinafter also referred to as the "short-culm grass plant of the present invention"). The short-culm grass plant of the present invention is, for example, a short-culm grass plant into which a mutation has been introduced at a position selected from the group consisting of 3,380,548 bases, 5,534,571 bases, and 5,538,047 bases from the end of the 5' short arm of chromosome 1. The mutation may be, for example, a single-nucleotide mutation. For example, the mutation may be selected from the group consisting of a T to C, G, or A substitution at 3,380,548 bases from the end of the 5' short arm of chromosome 1, a C to G, A, or T substitution at 5,534,571 bases from the end of the 5' short arm of chromosome 1, and a C to T, G, or A substitution at 5,538,047 bases from the end of the 5' short arm of chromosome 1, compared to the genomic sequence of a grass family plant that does not have d64. Furthermore, the mutation may be selected from the group consisting of a T to C substitution at 3,380,548 bases from the end of the 5' short arm of chromosome 1, a C to G substitution at 5,534,571 bases from the end of the 5' short arm of chromosome 1, and a C to T substitution at 5,538,047 bases from the end of the 5' short arm of chromosome 1, compared to the genomic sequence of a grass family plant that does not have d64.

[0035] As a further example, the mutation introduced into the short-culm grass plant of the present invention may be a substitution, insertion, or deletion of a region of 2 to several tens of bases including a position selected from the group consisting of 3,380,548 bases, 5,534,571 bases, and 5,538,047 bases from the end of the 5' short arm of chromosome 1.

[0036] Mutation introduction may be performed by any method commonly used in the art. For example, it may be performed by hybridization or by genetic engineering methods. Mutation introduction by hybridization can be achieved, for example, by the method for producing short-culm grasses of the present invention. Genetic engineering methods include, but are not limited to, the Agrobacterium method, particle gun method, electroporation method, polyethylene glycol (PEG) method, genome editing, etc.

[0037] Furthermore, the short-culm grasses of the present invention may be short-culm grasses in which the expression of a gene comprising a region of one to several tens of bases, including a site selected from the group consisting of 3,380,548 bases, 5,534,571 bases, and 5,538,047 bases from the end of the 5' short arm of chromosome 1, is suppressed. Here, "suppression" refers to the absence of expression of the gene or a reduction in the level of expression compared to a control in which the expression of the gene is not suppressed. Gene expression can be suppressed by any method commonly used in the art, including, but not limited to, antisense methods, mutagenesis, and RNA interference.

[0038] The short-culm grasses of the present invention have the same panicle length and grain size as Koshihikari, but are shorter in height. Preferably, the short-culm grasses of the present invention also have an increased number of panicles. For example, the short-culm grasses of the present invention have a culm length that is about 10% to 30% shorter and an increased number of panicles by about 20% to 50% compared to Koshihikari. For example, the short-culm grasses of the present invention have a culm length that is about 3 cm to 10 cm shorter and an increased number of panicles by about 2.5 to 5.5 compared to Koshihikari. Therefore, the short-culm grasses of the present invention have excellent lodging resistance and productivity.

[0039] The short-culm grass plant of the present invention may have a short-culm gene other than d64 introduced into it in addition to d64. Examples of short-culm genes other than d64 include, but are not limited to, sd1 and d60. When a short-culm gene other than d64 is introduced into the short-culm grass plant of the present invention, the culm length is preferably further shortened.

[0040] The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. [Example]

[0041] Example 1: Detection of short culm gene d64 When the F2 hybrids of Koshihikari and Koganebare were bred and the culm length of all individuals was examined, a 1:3 segregation of Koganebare-type short-culm individuals and Koshihikari-type long-culm individuals was observed. Furthermore, when the F2 hybrids of Koshihikari sd1 and Isehikari, and the F2 hybrids of Koshihikari sd1 and Koganebare were bred, a double short-culm type even shorter than Koshihikari sd1 was bred. Furthermore, when the F2 hybrids of Isehikari and Koganebare were bred, no segregation of culm length was observed. Therefore, it was found that Isehikari and Koganebare share a common short-culm gene that is different from sd1. This short-culm gene was named "d64."

[0042] Koshihikari sd1 is a short-culm variety registered as "Hikari Shinseiki." It was developed by 14 backcrosses using a homozygous sd1 short-culm line as the primary parent and Koshihikari as the recurrent parent. This Koshihikari sd1 was also used in Examples 2 and 3.

[0043] Furthermore, a total of six backcrosses were performed using double-dwarf individuals isolated in the F2 cross between Koshihikari sd1 and Isehikari as the nodal parent, Koshihikari as the dwarf parent, and Koshihikari sd1 as the tetrad parent. Koshihikari sd1 (BC8F4) was used here. The results showed that in the BC6F2 cross between sd1 Koshihikari*4 / / Koshihikari*2 / [Koshihikari sd1 x Isehikari F2 double-dwarf type], sd1 homozygous short-culm individuals with culm lengths of approximately 47-51 cm, similar to those of sd1 Koshihikari, double-dwarf individuals with short culms of approximately 35-40 cm, and intermediate individuals segregated in a ratio of 19 sd1 homozygous short-culm individuals: 27 intermediate individuals: 14 double-dwarf individuals, conforming to the theoretical ratio (χ 2= 6.42, 0.01 < P < 0.025). Therefore, it was confirmed that the short culm gene d64 different from sd1确实存在 in Koshihikari.

Example

[0044] Example 2: Mapping of the short culm gene d64 (1) Breeding of isogenic lines into which the short culm gene d64 or the late-heading gene Hd16 was introgressed In the F2 of the cross between Koshihikari and Kinuhikari, the F2 of the cross between Koshihikari sd1 and Kinuhikari, and the F2 of the cross between Koshihikari sd1 and Isehikari, Kinuhikari-type short culm individuals, double short culm individuals derived from Kinuhikari, and double short culm individuals derived from Isehikari were selected, and six backcrosses were carried out with Koshihikari as the recurrent parent twice and Koshihikari sd1 as the recurrent parent four times so that the non-d64 part was replaced with the Koshihikari genome, and "Koshihikari d64-k", "Koshihikari sd1d64-k", and "Koshihikari sd1d64-i" with 99.2% replaced with the Koshihikari genome were bred. Furthermore, late-heading type individuals were selected from the F2 of the cross between Koshihikari and Isehikari, and six backcrosses to Koshihikari were carried out to breed "Koshihikari Hd16" with 99.2% replaced with the Koshihikari genome. Koshihikari Hd16 was about 14 days later-heading than Koshihikari.

[0045] (2) Whole genome analysis Genomic DNA was extracted from the leaves of four types of isogenic lines "Koshihikari d64-k", "Koshihikari sd1d64-k" and "Koshihikari sd1d64-i", and "Koshihikari sd1" having the genetic background of Koshihikari bred in (1) above. The extraction of genomic DNA was carried out according to a conventional method. The genomic DNA derived from each extracted line was fragmented with Nextera transposon so that the peak became about 500 bp while tagging, and after preparing a whole genome DNA library, it was decoded with Illumina HiSeq 2500 (manufactured by Illumina, Inc.).

[0046] The resulting reads were quality checked using FastQC, and adapter sequences and reads with a quality score below 20 were removed using Trimmomatic. The quality was then checked again using FastQC. The Koshihikari genome sequence, which had been separately prepared in-house, was used as the reference sequence for alignment using BWA or Bowtie2. Secondary alignments were removed using SAMtools, and duplicate sequences amplified by PCR during library construction were removed using Picard. Reads near deletions and insertions were then realigned using GATK Realigner Target Creator and GATK Indel Realigner. After variant calling using GATK Haplotype Caller, low-quality SNPs were removed and VCF files were generated. Thus, the frequency distribution of SNPs relative to the Koshihikari genome was obtained for each of the above lines.

[0047] We confirmed that sd1 is located in the region 38,267,149-38,270,233 bp from the end of the short arm of chromosome 1 of "Koshihikari sd1" (a missense SNP G→T at position 38,267,510). A cluster of SNPs was distributed between 36,977,336 and 38,863,193 bp surrounding sd1, while the remaining regions were nearly identical to the Koshihikari genome. Furthermore, in "Koshihikari d64sd1," sd1 was located in the region 38,267,149-38,270,233 bp from the end of the short arm of chromosome 1 (a missense SNP G→T at position 38,267,510), and a cluster of SNPs was distributed between 34,616,598 and 38,863,193 bp surrounding sd1. Figure 1 shows the SNP frequency distribution indicating the relative positions of sd1 and d64 on chromosome 1 in double short-culm Koshihikari (Koshihikari sd1d64-i) obtained by whole-genome analysis.

[0048] Based on the SNP frequency distribution across the Koshihikari genome in each line, we searched for SNPs that were absent in "Koshihikari Hd16" but specifically present in "Koshihikari d64-k," "Koshihikari sd1d64-k," and "Koshihikari sd1d64-i." Eight SNPs were identified on chromosomes 1, 4, 7, 9, and 10. Furthermore, by aligning these SNPs with the Nipponbare genome, we searched for SNPs shared with Nipponbare. Nipponbare is a short-culm variety that is the parent of Ogonbare, and its entire genome sequence has been published. As a result, the SNPs specific to the d64 isogenic line originating from Nipponbare were narrowed down to three locations at the terminal end of the short arm of chromosome 1 and one location on chromosome 7.

[0049] Of these, the three SNPs at the end of the short arm of chromosome 1 were located at 3,380,548 bases, 5,534,571 bases, and 5,538,047 bases from the 5' end of the short arm of chromosome 1. It was found that mutations of "T to C" at 3,380,548 bases from the 5' end of the short arm, "C to G" at 5,534,571 bases, and "C to T" at 5,538,047 bases, respectively, occurred between Koshihikari and the d64 isogenic line.

[0050] (3) Identification of the d64 locus by linkage analysis We investigated the linkage between known SSR markers near the candidate SNPs and the Ogonharu type short culm.

[0051] Leaf samples from short-culm (d64 homozygous) individuals isolated from Koshihikari × Koganeharu F2 were placed in a 2 mL CK28 grinding tube containing ceramic beads (YTZ balls) and frozen in liquid nitrogen for 1 minute. The samples were then ground using a Precellys 24 (Bertin Instruments) high-speed bead-type cell disrupter at 6500 rpm for 20 seconds each, with a 120-second interval. 500 μL of CTAB (cetyltrimethylammonium bromide) was added to the tube and incubated at 55°C for 30 minutes. 500 μL of chloroform / isoamyl alcohol (24:1) was added, shaken for 10 minutes, and then centrifuged at 5000 rpm for 10 minutes at 4°C. The supernatant was then transferred to a new 1.5 mL tube sterilized by autoclaving. 50 μL of 3 M sodium acetate (pH 7.8) and 500 μL of isopropanol were added to the tube to aggregate DNA. This was centrifuged at 5000 rpm at 4° C. for 10 minutes to precipitate the mixture, the supernatant was discarded, and 300 μl of 70% ethanol was added, followed by centrifugation at 5000 rpm at 4° C. for 10 minutes. After centrifugation, the supernatant was discarded, the mixture was air-dried, and 100 μl of sterilized pure water was added.

[0052] PCR reactions were performed using the DNA samples extracted as described above. The PCR program consisted of an initial heat denaturation at 95°C for 2 minutes, followed by 35 cycles of heat denaturation at 95°C for 30 seconds, annealing at 52°C for 30 seconds, and extension at 72°C for 30 seconds, followed by a 5-minute hold at 72°C. Linkage analysis was performed at three sites using the primer set PM10132, RM10183, and RM3035. The primer sets are listed in Table 1. PM10132 is an SSR marker located 2.525 Mb from the end of the short arm of chromosome 1. RM10183 is an SSR marker located 3.745 Mb from the end of the short arm of chromosome 1. RM3035 is an SSR marker located 5.151 Mb from the end of the short arm of chromosome 1.

[0053] [Table 1]

[0054] The PCR product was loaded into the gel cartridge of the QIAxcel DNA Screening kit (2400) of the QIAxcel DNA / RNA capillary electrophoresis system (QIAGEN). The alignment marker was QX Alignment Marker 15 / 1 kb, and the DNA size marker was QX DNA Size Marker pUC18 / Haelll. The electrophoresis conditions were sample injection at 5 kV for 10 seconds, followed by electrophoresis at 5 kV for 320–420 seconds.

[0055] Linkage analysis revealed that the recombination value was 40% in RM10132, 7.1% in RM10183, and 45% in RM3035, with particularly strong linkage in RM10183. Therefore, it was confirmed that of the candidate SNPs obtained in (2), three SNPs at the end of the short arm of chromosome 1, particularly the SNP at 3,380,548 bases from the 5' end of the short arm of chromosome 1, are involved in d64. [Example]

[0056] Example 3: Characteristics of rice plants carrying the short culm gene d64 "Koshihikari d64-k," d64, and "Koshihikari sd1d64-i," as well as "Koshihikari" and "Koshihikari sd1," obtained in Example 2, were cultivated for 45 days, and the number of panicles and main culms of each individual were examined, and the panicle length, culm length, and length of each internode were measured. The results are shown in Tables 2-1 to 2-3 and Figure 2. Each cultivated individual is also shown in Figure 3.

[0057] [Table 2-1]

[0058] [Table 2-2]

[0059] [Table 2-3]

[0060] As shown in Tables 2-1 to 2-3, "Koshihikari d64," which was introgressed with d64 in the Koshihikari genetic background, had a culm length 10.4 cm (16.8%) shorter and stronger than Koshihikari. The double-dwarf-culm "Koshihikari sd1d64" had a culm length 30.6 cm (49.5%) shorter and stronger than Koshihikari. Furthermore, "Koshihikari d64" had an increased number of panicles (from 8.6 to 12.4, a 44.1% increase) compared to Koshihikari. Furthermore, "Koshihikari d64" had a culm length 5.0 cm (10.6%) longer, a second internode length 3.9 cm (30.9%) longer, and an increased number of panicles (from 9.5 to 12.4, a 30.5% increase) compared to "Koshihikari sd1" with sd1. Therefore, it was found that d64 has lodging resistance due to short culms and has superior productivity to sd1. [Industrial Applicability]

[0061] According to the present invention, the locus of the novel short-culm gene d64 and the causative SNP have been identified. Therefore, based on the d64 DNA marker, short-culm grasses can be identified and produced without relying on conventional short-culm genes. Grass plants carrying d64 have normal panicle length and grain size, but are shorter in height, and have an increased number of panicles, which gives them not only lodging resistance but also high yielding potential. Therefore, the present invention can be used in the agricultural field for rice production and for the breeding of various grasses. [Sequence List Free Text]

[0062] SEQ ID NO:1; Primer SEQ ID NO:2; Primer SEQ ID NO:3; Primer SEQ ID NO:4; Primer SEQ ID NO:5; Primer SEQ ID NO:6; Primer

Claims

1. A method for selecting short-culm grasses, comprising determining the presence of a short-culm gene by detecting a DNA marker present in a region 3.3 to 5.6 Mb from the end of the 5' short arm of chromosome 1 of the grasses, wherein the DNA marker is RM10183 or a SNP located at a position selected from the group consisting of 3,380,548 base positions, 5,534,571 base positions, and 5,538,047 base positions from the end of the 5' short arm of chromosome 1 of the grasses.

2. 2. The method of claim 1, wherein the SNP is selected from the group consisting of a T to C substitution at base position 3,380,548, a C to G substitution at base position 5,534,571, and a C to T substitution at base position 5,538,047.

3. A method for producing a short-culm grass plant, comprising: (a) crossing a first parent grass plant having a short culm trait with a second parent grass plant to obtain a hybrid plant; (b) crossing the hybrid plants with each other, or backcrossing or multi-line crossing with the hybrid plants to obtain progeny plants; (c) furthermore, crossing between progeny plants, or backcrossing or multi-line crossing with progeny plants may be repeatedly performed; (d) selecting short-culm grasses for any or all generations of plants by the method of claim 1 or 2. A method comprising:

4. 4. The method of claim 3, wherein the first parent grass plant is a short-culm grass plant containing a mutation at one or more positions selected from the group consisting of 3,380,548 base positions, 5,534,571 base positions, and 5,538,047 base positions from the 5' end of the short arm of chromosome 1.

5. 5. The method of claim 4, wherein the mutation is selected from the group consisting of a T to C substitution at base position 3,380,548, a C to G substitution at base position 5,534,571, and a C to T substitution at base position 5,538,047.

6. The method according to any one of claims 3 to 5, wherein the second parent grass plant is a grass plant that does not have the trait of short culm.

7. A short-culm grass plant having a mutation introduced into a position selected from the group consisting of 3,380,548 base positions, 5,534,571 base positions, and 5,538,047 base positions from the 5' short arm end of chromosome 1.

8. 8. The short-culm grass plant of claim 7, wherein the mutation is selected from the group consisting of a T to C substitution at base position 3,380,548, a C to G substitution at base position 5,534,571, and a C to T substitution at base position 5,538,047.

9. Use of a DNA marker for determining the presence of a short-culm gene in a grass plant and / or for selecting a short-culm grass plant, wherein the DNA marker is RM10183 or a SNP at a position selected from the group consisting of 3,380,548 base positions, 5,534,571 base positions, and 5,538,047 base positions from the end of the 5' short arm of chromosome 1 of the grass plant.

10. 10. The use of claim 9, wherein the SNP is selected from the group consisting of a T to C substitution at base position 3,380,548, a C to G substitution at base position 5,534,571, and a C to T substitution at base position 5,538,047.

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