Nucleic acid for controlling number of florets per spikelet

Abstract

The present invention provides a method for detecting that a target gramineous plant is a gramineous plant producing two florets per spikelet, wherein the method includes a step for confirming the presence of a recombination site due to inversion of the base sequence in the 5'-side region of the WUSCHEL gene, and the 5'-side sequence of the AUXIN RESPONSE FACTOR 5 gene is adjacent to the recombination site.

Description

Nucleic acids controlling the number of florets per spikelet

[0001] The present invention relates to a nucleic acid that controls the number of florets per spikelet, and a method for detecting a grass plant that produces two florets per spikelet.

[0002] Grass plants such as sorghum are highly valuable crops that can be used for a variety of purposes, such as livestock feed and biofuel production. In particular, grains of grasses can be used as food, so increasing grain yields is extremely important.

[0003] Grains are produced from florets contained in spikelets, and many grass crops, such as rice, barley, maize, and sorghum, have one floret per spikelet. Recently, a gene, GNI1, that controls the number of florets in wheat has been identified (Non-Patent Document 1). Sorghum with two florets per spikelet has also been reported (Non-Patent Document 2).

[0004] S. Sakuma et al. , Unleasing floret fertility in wheat through the mutation of a homeobox gene. PNAS, 116(11), 5182-5187 (2019). ZHOU Shi-chen et al. , Genetic Analysis and Gene Mapping of Sorghum Double-grain Mutant Dgs. Biotechnology Bulletin, 38(7), 171-177 (2022).

[0005] The present inventors believed that the ability to select grasses with an increased number of florets per spikelet would facilitate the breeding of grasses with increased grain yield. However, the factors that cause grasses to produce two florets per spikelet have not been elucidated. The problem that the present invention aims to solve is to advance the elucidation of these factors and to provide a method for detecting grasses that produce two florets per spikelet.

[0006] As a result of extensive research to achieve the above object, the present inventors have discovered a novel nucleic acid that controls the number of florets per spikelet. Furthermore, they have found that by confirming the presence of a recombination site due to inversion of a nucleotide sequence in the 5' region of the WUSCHEL gene (also referred to herein as WUS), which is adjacent to the 5' sequence of the AUXIN RESPONSE FACTOR 5 gene (also referred to herein as ARF5), it is possible to detect a grass plant that produces two florets per spikelet.

[0007] That is, the present application encompasses the following inventions: [1] A method for detecting that a target grass plant is a grass plant that produces two florets per spikelet, the method comprising a step of confirming the presence of a recombination site due to a nucleotide sequence inversion in the 5' region of the WUSCHEL gene, wherein the recombination site is adjacent to the 5' sequence of the AUXIN RESPONSE FACTOR 5 gene. [2] The method according to [1], wherein the nucleotide sequence inversion is an inversion of a double-stranded sequence containing the AUXIN RESPONSE FACTOR 5 gene. [3] The method according to [1] or [2], wherein the step of confirming the presence of a recombination site is a step of obtaining an amplification product containing the 5' flanking sequence and the 3' flanking sequence of the recombination site, and the length of the amplification product is 10,000 bp or less. [4] The method according to [3], wherein the amplification product comprises the nucleotide sequence from bases 810 to 830 on the 5' side of the WUSCHEL gene and the nucleotide sequence from bases 1620 to 1640 on the 5' side of the AUXIN RESPONSE FACTOR 5 gene. [5] A nucleic acid comprising a nucleotide sequence having at least 80% identity with the nucleotide sequence shown in SEQ ID NO: 1, the nucleic acid controlling the number of florets per spikelet. [6] A nucleic acid consisting of the nucleotide sequence shown in SEQ ID NO: 1. [7] A vector comprising the nucleic acid according to [5] or [6]. [8] A transformant having the nucleic acid according to [5] or [6]. [9] The transformant according to [8], wherein the transformant is a plant.

[10] The transformant according to [9], wherein the plant is sorghum.

[0008] The present invention can provide a method for detecting a grass plant that produces two florets per spikelet, and can also provide a nucleic acid that controls the number of florets per spikelet.

[0009] The F was obtained by crossing a general sorghum line that produces one grain per spikelet (called the Single parent line) with a sorghum line that produces two grains per spikelet (called the Twin parent line) found in a germplasm population. １ The results of examining the phenotype of the individual are shown below. １ F obtained by self-fertilization of individuals ２ The results of QTL analysis using the population are shown. ２The results of investigating the relationship between the genotype of the nearest marker for a QTL near 59 Mb in the genome of each individual in the population and grain traits are shown. Using the sorghum genome as an example, the relative positions of each gene due to inversion of the base sequence are shown. In typical sorghum, WUS and ARF5 are positioned on the chromosome as shown for a single parent line. Therefore, in a single parent line, the 5'-side regions of WUS and ARF5, which are considered to be the respective promoter regions, are aligned with WUS, the 5'-side region of WUS, ARF5, and the 5'-side region of ARF5 on the chromosome. In other words, the 3'-side region of ARF5 is adjacent to the 5'-side region of WUS. However, in the Twin parent line, the double-stranded sequence containing ARF5 and the 5' sequence of ARF5 is inverted (rotated 180°) in the 5' region of WUS, resulting in the following arrangement on the chromosome: WUS, the 5' region of WUS, the 5' region of ARF5, and ARF5. In other words, in the Twin parent line, the 5' region of WUS and the 5' region of ARF5 are adjacent. Therefore, the Twin parent line contains a recombination site due to the inversion of the nucleotide sequence. A single plant was transformed with a vector containing the sequence shown in SEQ ID NO: 1, and spikelets were observed after heading and before flowering. It was confirmed that the number of florets in the spikelet increased from the original one to two (indicating the trait of two florets / spikelet).

[0049] Figure 1 shows the results of transforming a single individual with a vector containing the sequence shown in SEQ ID NO: 1 in which the ARF5 function has been disrupted, or a vector in which the WUS function has been disrupted, and observing the spikelets after heading and before flowering. Transformants into which the former vector has been introduced exhibit the 2 floret / spikelet trait, similar to transformants into which the unmodified vector has been introduced, while transformants into which the latter vector has been introduced exhibit the original 1 floret / spikelet trait before transformation. Primers I to III (SEQ ID NOs: 2 to 4) were used to generate F1 clones obtained by crossing a Single parent line (a Single parent line homozygous individual), a Twin parent line (a Twin parent line homozygous individual), and a F2 clone obtained by crossing the two. １ The results of PCR using DNA extracted from an individual (heterozygous individual) as a template are shown.

[0010] Hereinafter, an embodiment of the present invention (hereinafter referred to as "the present embodiment") will be described, but the scope of the present invention should not be interpreted as being limited to the following embodiment.

[0011] The present embodiment will be specifically described below by showing an embodiment, but the present invention is not limited to this embodiment.

[0012] As a first aspect, this embodiment provides a method for detecting that a target grass plant is a grass plant that produces two florets per spikelet, the method comprising a step of confirming the presence of a recombination site due to a nucleotide sequence inversion in the 5' region of the WUSCHEL gene, wherein the recombination site is adjacent to the 5' sequence of the AUXIN RESPONSE FACTOR 5 gene.

[0013] Examples of grasses that can be subjected to the method of this embodiment include sorghum, Erianthus, Giant Miscanthus, Napier grass, switchgrass, maize, sugarcane, pearl millet, millet, finger millet, foxtail, bamboo, rice, barnyard millet, foxtail millet, lawn grass, barley, wheat, Miscanthus sinensis, and pontiffin. Among these, grasses known to have one floret per spikelet in the wild, such as sorghum, rice, barley, and maize, are preferred, with sorghum being more preferred. Here, grasses with one floret per spikelet may have a gene that suppresses the development or differentiation of two or more florets per spikelet. In this embodiment, a grass that produces two florets per spikelet may be a mutant strain of a grass that has one floret per spikelet in the wild. In one embodiment, the present embodiment also provides a method for detecting that a target grass plant having one floret per spikelet has been mutated into a grass plant having two florets per spikelet.

[0014] As used herein, the term "spikelet" refers to a plant organ that is a unit constituting an inflorescence and has a structure in which one or more florets are surrounded by glumes.

[0015] As used herein, the term "floret" refers to a unit constituting an inflorescence, an organ including glumes, lemma, scales, stamens, pistils, etc., and is included in a spikelet.

[0016] As used herein, "WUSCHEL gene" or "WUS" generally refers to a gene involved in maintaining the undifferentiated state of cells in the shoot apical meristem, the plant hormone signal transduction system, and the like.

[0017] As used herein, "AUXIN RESPONSE FACTOR 5 gene" or "ARF5" generally refers to a transcription factor that controls the transcription of auxin responsive genes.

[0018] Both WUS and ARF5 are involved in meristem regulation.

[0019] As used herein, "inversion of a base sequence" refers to the fusion of fragments generated by two cleavage sites in a base sequence at the two cleavage sites in the reversed orientation, specifically, rotated 180°. In this embodiment, the inversion of a base sequence refers to the inversion of a double-stranded sequence.

[0020] As used herein, "recombination site" refers to a site where cleavage in an inverted nucleotide sequence and fusion of the cleaved fragments occurs, or a site where cleavage in an inverted nucleotide sequence and fusion of the cleaved fragments may occur. The recombination site confirmed to exist in the method of this embodiment is the recombination site after inversion. The recombination site may be a portion between the cleaved and fused nucleic acids. Furthermore, since cleavage in an inversion involves cleavage of phosphodiester bonds between adjacent nucleic acids, the recombination site may be a portion containing a phosphodiester bond between the cleaved and fused nucleic acids. Furthermore, since the inversion of a nucleotide sequence in this embodiment is an inversion of a double-stranded sequence, cleavage occurs in both strands at the inversion. Therefore, the recombination site in this embodiment exists in both strands. The presence of a recombination site in both strands means that one strand also has a recombination site at a position corresponding to the position of the recombination site in the other strand. The positional relationship of the recombination sites in both strands may be such that blunt ends are generated when cleavage occurs along the recombination sites in both strands.

[0021] The method of this embodiment includes a step of confirming the presence of a recombination site due to a nucleotide sequence inversion in the 5' region of WUS. The genome of a grass plant that produces two florets per spikelet (also referred to as a "Twin individual" herein) has an inverted nucleotide sequence in the 5' region of WUS. Therefore, by confirming the recombination site due to an inversion in the 5' region of WUS, it is possible to detect whether the individual is a Twin individual. In this embodiment, the inversion in the Twin individual is an inversion of a double-stranded sequence containing ARF5. Furthermore, in this embodiment, the double-stranded sequence containing ARF5 also contains the 5' sequence of ARF5.

[0022] As used herein, the "5' side of gene X" or "3' side of gene X" refers to the 5' or 3' region of gene X on the sense strand of gene X. Preferably, the 5' side of gene X refers to the 5' side of the start point of the coding region of gene X, and the 3' side of gene X refers to the 3' side of the end point of the coding region of gene X. The 5' region of gene X may also include the 3' region of gene X on the antisense strand of gene X, and the 3' region of gene X may also include the 5' region of gene X on the antisense strand of gene X.

[0023] The genome of a typical grass plant with one floret per spikelet (also referred to herein as a "single individual") without inversion has ARF5 on the 5' side of WUS (the sense strand of WUS and the sense strand of ARF5 are the same strand). On the other hand, in the genome of a Twin individual, the double-stranded sequence containing ARF5 and the 5' sequence of ARF5 is inverted (rotated 180°) in the 5' region of WUS, so WUS, ARF5, and the 5' sequence of ARF5 are on different strands (the sense strand of WUS and the sense strand of ARF5 are different strands). Furthermore, while two recombination sites are generated in the genome due to the inversion, the recombination site confirmed by the method of this embodiment is the recombination site near the 5' region of WUS. Therefore, the recombination site confirmed by the method of this embodiment is adjacent to the 5' sequence of ARF5. In other words, the 5' sequence of ARF5 is adjacent to the 3' side of the recombination site near the 5' region of WUS in the antisense strand of WUS. Figure 4 shows the positional relationship of each gene due to inversion of the base sequence, using the sorghum genome as an example.

[0024] The position at which the inversion occurs in the Twin individual varies depending on the type of grass family plant that is the target of the method of this embodiment, etc.; for example, when the target grass family plant is sorghum, the nucleic acid between the 800th to 850th bases on the 5' side of WUS and the nucleic acid adjacent to these nucleic acids, and the nucleic acid between the 1620th to 1670th bases on the 5' side of ARF5 and the nucleic acid adjacent to these nucleic acids are cleaved, and the cleaved fragments are fused in the reverse direction, resulting in the inversion. Since the recombination site confirmed in this embodiment is a recombination site near the WUS, in one embodiment, this embodiment provides a method for detecting that a target grass plant is a grass plant that produces two florets per spikelet, the method comprising the step of confirming the presence of a recombination site located between the nucleic acid at position 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, or 840 5' of the WUS and the nucleic acid adjacent to that nucleic acid. In one embodiment, the present embodiment provides a method for detecting that a sorghum of interest is a sorghum that produces two florets per spikelet, the method comprising the step of confirming the presence of a recombination site located between the nucleic acid at position 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, or 840 5' of the WUS and a nucleic acid adjacent to that nucleic acid.

[0025] Twin individuals may express a trait of producing two florets per spikelet by inverting the base sequence in the 5' region of WUS and acquiring a new promoter sequence in WUS. In Twin individuals, the gene that determines the trait of producing two florets per spikelet may be WUS. Furthermore, the trait of producing two florets per spikelet may be dominant, and when the gene that determines the trait of producing two florets per spikelet is WUS, WUS may be a dominant gene. When the trait of producing two florets per spikelet is a dominant gene, there exist homozygous Twin individuals and heterozygous Twin individuals.

[0026] A method for confirming the presence of a recombination site can be appropriately determined by those skilled in the art. For example, confirmation may be made by obtaining an amplification product of a region containing the recombination site and analyzing the amplification product, or by directly sequencing the region. However, confirmation by analyzing the amplification product is preferred. The amplification product contains the 5'-flanking sequence and the 3'-flanking sequence of the recombination site in a single individual or a twin individual. The recombination site in an amplification product of a single individual refers to a site where cleavage at the inversion of the nucleotide sequence and fusion of the cleaved fragments may occur, or a site corresponding to a site where inversion of the nucleotide sequence occurs in a twin individual. When the grass family plant to be detected by the method of this embodiment is a twin individual, the recombination site in the genome of the grass family plant is adjacent to the 5'-side sequence of ARF5, i.e., the 5'-side sequence of ARF5 is adjacent to the 5'-side region of WUS, and therefore the amplification product of the region containing the recombination site obtained from a twin individual contains the 5'-side sequence of ARF5. On the other hand, a similar amplification product obtained from a single individual does not contain a recombination site and does not contain the 5'-side sequence of ARF5 adjacent to the 5'-side region of WUS. Therefore, by analyzing the sequence and length of the amplification product of the region containing the recombination site, the presence of a recombination site due to inversion can be confirmed, and it can be detected that the plant is a grass family plant that produces two florets per spikelet. In the method of this embodiment, the step of confirming the presence of a recombination site due to nucleotide sequence inversion in the 5'-side region of WUS may be a step of obtaining an amplification product containing the 5'-side flanking sequence and the 3'-side flanking sequence of the recombination site, a step of analyzing an amplification product containing the 5'-side flanking sequence and the 3'-side flanking sequence of the recombination site, or a step of confirming the presence of a recombination site by analyzing an amplification product containing the 5'-side flanking sequence and the 3'-side flanking sequence of the recombination site.

[0027] When the presence of a recombination site is confirmed by analyzing the amplification product, the amplification product obtained from the Twin individual contains a sequence between the WUS and the recombination site, which is adjacent to the recombination site near the WUS confirmed by the method of this embodiment, and a sequence on the 5' side of ARF5 between the ARF5 and the recombination site, which is adjacent to the recombination site near the WUS.

[0028] The sequence between the WUS and the recombination site contained in the amplification product obtained from the Twin individual may be any sequence as long as it is adjacent to the recombination site near the WUS confirmed by the method of this embodiment. For example, when the grass plant targeted by the method of this embodiment is sorghum, the sequence between the WUS and the recombination site contained in the amplification product is preferably a sequence contained in the region from bases 1 to 840 on the 5' side of the WUS. In one embodiment, the present embodiment provides a method for detecting that a target grass plant is a grass plant that produces two florets per spikelet, the method comprising the step of obtaining an amplification product comprising a sequence located at bases 100 to 830, 200 to 830, 300 to 830, 400 to 830, 500 to 830, 600 to 830, 700 to 830, 800 to 830, 810 to 830, 820 to 830, or 410 to 834 on the 5' side of WUS. In one embodiment, the present embodiment provides a method for detecting that a target sorghum is a sorghum that produces two florets per spikelet, the method comprising the step of obtaining an amplification product comprising the sequence of bases 100 to 830, 200 to 830, 300 to 830, 400 to 830, 500 to 830, 600 to 830, 700 to 830, 800 to 830, 810 to 830, 820 to 830, or 410 to 834 on the 5' side of the WUS.

[0029] As used herein, "the Yth position on the 5' side of gene X" refers to the Yth position of a nucleic acid in the sense strand of gene X, when the position of the nucleic acid adjacent to the 5' side of the nucleic acid that is the start point of the coding region of gene X is counted as the first position.

[0030] The 5'-side sequence of ARF5 between ARF5 and the recombination site contained in the amplification product obtained from Twin individuals may also be any sequence as long as it is adjacent to the recombination site near WUS. For example, when the grass family plant targeted by the method of this embodiment is sorghum, the sequence contained in the amplification product between ARF5 and the recombination site is preferably a sequence contained in the region from bases 1 to 1650 on the 5' side of ARF5. In one embodiment, this embodiment provides a method for detecting that a target grass plant is a grass plant that produces two florets per spikelet, the method comprising the step of obtaining an amplification product comprising a sequence located at bases 100 to 1640, 500 to 1640, 800 to 1640, 1000 to 1640, 1200 to 1640, 1400 to 1640, 1600 to 1640, 1620 to 1640, 1630 to 1640, or 1010 to 1645 on the 5' side of ARF5. In one embodiment, this embodiment provides a method for detecting that a target sorghum is a sorghum that produces two florets per spikelet, the method comprising the step of obtaining an amplification product comprising a sequence located at bases 100 to 1640, 500 to 1640, 800 to 1640, 1000 to 1640, 1200 to 1640, 1400 to 1640, 1600 to 1640, 1620 to 1640, 1630 to 1640, or 1010 to 1645 on the 5' side of ARF5.

[0031] In one embodiment, the present embodiment provides a method for detecting whether a target grass plant is a grass plant that produces two florets per spikelet, the method comprising the step of obtaining an amplification product comprising the sequence from bases 810 to 830 on the 5' side of WUS and the sequence from bases 1620 to 1640 on the 5' side of ARF5. In one embodiment, the present embodiment provides a method for detecting whether a target sorghum is a sorghum that produces two florets per spikelet, the method comprising the step of obtaining an amplification product comprising the sequence from bases 810 to 830 on the 5' side of WUS and the sequence from bases 1620 to 1640 on the 5' side of ARF5. In one embodiment, the present embodiment provides a method for detecting that a target sorghum is a sorghum that produces two florets per spikelet, the method comprising the step of obtaining an amplification product comprising the sequence from bases 410 to 834 on the 5' side of WUS and the sequence from bases 1010 to 1645 on the 5' side of ARF5.

[0032] Furthermore, when the presence of a recombination site is confirmed by analyzing the amplification product, the amplification product is preferably 10,000 bp or less. The length of the amplification product is preferably 2,500 bp or less, and may be, for example, 500 bp to 2,000 bp, 500 bp to 1,500 bp, 500 bp to 1,200 bp, 700 bp to 2,000 bp, 700 bp to 1,500 bp, or 700 bp to 1,200 bp. In one embodiment, this embodiment provides a method for detecting that a target sorghum is a grass plant that produces two florets per spikelet, the method comprising the step of obtaining an amplification product of 10,000 bp or less. In one embodiment, the present embodiment provides a method for detecting that a target sorghum is a grass plant that produces two florets per spikelet, the method comprising a step of obtaining an amplification product having a length of 700 bp or more and 1200 bp or less.

[0033] An example of a method for confirming the presence of a recombination site by analyzing an amplification product is as follows: Using the genomic DNA of a target grass plant as a template, an amplification product is obtained using Primer I, which anneals to an arbitrary region between the recombination site near WUS and WUS in Twin individuals and Single individuals, Primer II, which anneals to an arbitrary region between the recombination site near WUS and ARF5 in the complementary strand to the strand to which Primer I anneals in Twin individuals, and Primer III, which anneals to an arbitrary region between the recombination site near WUS and ARF5 in the complementary strand to the strand to which Primer I anneals in Single individuals. If the target grass plant is a twin individual, an amplification product is obtained using primers I and II, if the target grass plant is a single individual, an amplification product is obtained using primers I and III, and if the twin individual is heterozygous and the trait of double grains is dominant, two types of amplification product are obtained: one using primers I and II and one using primers I and III. Because the amplification product using primers I and II and the amplification product using primers I and III differ in sequence and length, by analyzing the amplification products by electrophoresis, sequencing, or the like, it is possible to detect that the target grass plant is a twin individual, i.e., a grass plant that produces two florets per spikelet. Furthermore, by analyzing the amplification products by the Restriction Fragment Length Polymorphism (RFLP) method or Southern hybridization method, it is possible to detect that the target grass plant is a grass plant that produces two florets per spikelet. When analyzing by the RFLP method or Southern hybridization method, it is possible to detect that the target grass plant is a grass plant that produces two florets per spikelet, for example, based on the difference in fragment lengths of the amplification products cleaved with a restriction enzyme.

[0034] The aspects of the method provided as the first embodiment also apply to other embodiments, such as the second embodiment described below.

[0035] As a second embodiment, this embodiment provides a nucleic acid that controls the number of florets per spikelet, the nucleic acid comprising a base sequence having at least 80% identity to the base sequence shown in SEQ ID NO: 1. The nucleic acid that controls the number of florets per spikelet may be a nucleic acid that causes two florets to be produced per spikelet.

[0036] SEQ ID NO: 1 is an example of a sequence including WUS, ARF5, and a region between WUS and ARF5 in the sorghum genome sequence.

[0037] As used herein, "controlling the number of florets per spikelet" refers to determining the number of florets contained in a spikelet, and the protein encoded by the nucleic acid of this embodiment has the activity of determining the number of florets contained in a spikelet. The nucleic acid of this embodiment that controls the number of florets per spikelet may be a nucleic acid that encodes a protein that has the activity of controlling the number of florets per spikelet. This embodiment also provides a protein that is encoded by a nucleic acid comprising a nucleotide sequence having at least 80% identity to the nucleotide sequence shown in SEQ ID NO: 1, and that has the activity of controlling the number of florets per spikelet.

[0038] As used herein, "nucleic acid" refers to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or chimeric nucleic acid thereof, and may be an artificially synthesized nucleic acid, and may be a single-stranded nucleic acid or a double-stranded nucleic acid. Nucleic acids may also contain modified nucleotides. Nucleic acids may be used interchangeably with oligonucleotides and polynucleotides.

[0039] The nucleic acid of this embodiment comprises a base sequence having at least 80% identity with the base sequence shown in SEQ ID NO:1.

[0040] As used herein, "identity" refers to the percentage (%) of nucleotides that are identical in the same position or in the same row when two base sequences are aligned.

[0041] For example, a base sequence having 80% identity with a specific base sequence consisting of 100 nucleotides may be a base sequence in which, when aligned with the specific base sequence consisting of 100 nucleotides, there are 80 locations where identical nucleotides are lined up in the same position or row.

[0042] In one embodiment, the nucleic acid of this embodiment is a nucleic acid comprising a base sequence having 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to the base sequence shown in SEQ ID NO: 1. In one embodiment, the nucleic acid of this embodiment is a nucleic acid consisting of the base sequence shown in SEQ ID NO: 1.

[0043] In one embodiment, the nucleic acid of this embodiment is a nucleic acid comprising a base sequence comprising 1 to 2630, 1 to 2500, 1 to 2000, 1 to 1500, 1 to 1000, 1 to 500, 1 to 100, or 1 to 50 nucleotide additions, substitutions, and / or deletions in the base sequence shown in SEQ ID NO: 1. The nucleic acid of this embodiment may also be a nucleic acid comprising a base sequence comprising 1 to 20, 1 to 10, 1 to 5, 1 to 3, 1 to 2, or 1 nucleotide additions, substitutions, and / or deletions per 100 nucleotides in the base sequence shown in SEQ ID NO: 1. The upper limit of 1 to 20 nucleotides may be 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1. In one embodiment, the nucleic acid of this embodiment is a nucleic acid that controls the number of florets per spikelet. Herein, when described as "X to Y (X and Y are 0 or positive numbers)," X to Y are synonymous with X or more and Y or less, and indicate a range that includes the values ​​of X and Y.

[0044] The nucleic acid in this embodiment may be contained in a vector. The type of vector is not particularly limited, and any vector known to those skilled in the art may be used, for example, a plasmid, a cosmid, an episome, an artificial chromosome, a phage, a viral vector, etc. When the nucleic acid is contained in a vector, the nucleic acid may be incorporated into the vector together with surrounding sequences of the nucleic acid and factors necessary for transcription and translation, such as a promoter, an enhancer, a terminator, etc., and the nucleic acid may exist contiguous or discontinuous with these in the vector. Here, as used herein, the term "vector" refers to a concept that includes cloning vectors and expression vectors, and refers to a nucleic acid that carries a gene of interest so as to transform a host, preferably a cell, and promote the expression (e.g., transcription and translation) of the introduced sequence.

[0045] This embodiment also provides a transformant into which the nucleic acid or vector has been introduced. As used herein, "transformation" refers to introducing a nucleic acid or vector into a cell or the like so that the cell produces a peptide of interest or expresses a trait of interest. The transformant in this embodiment may be in any form as long as it expresses a protein having the activity of controlling the number of florets in spikelets, but is preferably a cell (host cell), a cell cluster, a tissue, or a tissue cluster. Specifically, the cell may be a plant cell or the like, the cell cluster may be a callus or the like, the tissue may be a seed or the like, and the tissue cluster may be a plant or the like. This embodiment also provides a plant cell, callus, seed, or plant as a transformant. The plant is preferably a grass plant, more preferably sorghum. When the transformant in this embodiment is a host cell, the host cell may contain a vector expressing an endonuclease in addition to the vector containing the nucleic acid of this embodiment. The protein having the activity of controlling the number of florets in spikelets in the transformant may be expressed from the introduced nucleic acid or vector, or from a genome recombined by the nucleic acid or vector. The seeds and plants as the transformants of this embodiment may be those into which the nucleic acid or vector of this embodiment has been directly introduced, or may be those differentiated from the callus as the transformant of this embodiment.Furthermore, the plants as the transformants of this embodiment may be those which have been grown from the seeds as the transformants of this embodiment.

[0046] The nucleic acid, vector, and transformant of this embodiment may be produced by any method known to those skilled in the art, and the produced nucleic acids, vectors, and transformants may be used to produce a protein having activity to control the number of florets in a spikelet, or a plant having the protein. Furthermore, the nucleic acid of this embodiment may be a nucleic acid having a nucleotide sequence complementary to a nucleotide sequence having at least 80% identity to the nucleotide sequence set forth in SEQ ID NO: 1, and the nucleic acid may also be produced by any method known to those skilled in the art.

[0047] The protein of this embodiment having the activity of controlling the number of florets per spikelet may also be produced by any method known to those skilled in the art. For example, it may be produced by introducing the nucleic acid of this embodiment into a host, transforming it, and allowing it to be expressed in the host, or it may be produced by extracting it from a plant that has been modified to contain the nucleic acid of this embodiment.

[0048] When a plant, preferably a grass family plant, more preferably sorghum, contains the nucleic acid of this embodiment, the number of florets contained in the spikelets of the plant increases compared to a plant that does not contain the nucleic acid of this embodiment. For example, when a plant, preferably a grass family plant, more preferably sorghum, which has one floret in its spikelets, contains the nucleic acid of this embodiment, the number of florets in the spikelets increases to two. Increasing the number of florets in the spikelets can increase the grain yield or the number of seeds obtained per plant. The position at which the nucleic acid of this embodiment is contained in the plant may be arbitrary as long as the number of florets contained in the spikelets in the plant is controlled by the nucleic acid, but it is preferable that the genome of all cells in the plant contain the nucleic acid of this embodiment.

[0049] When the number of florets in spikelets of a plant containing a nucleic acid of this embodiment is greater than that of a plant not containing the nucleic acid, it can be evaluated that the nucleic acid is capable of controlling the number of florets per spikelet. For example, spikelets of a plant containing a nucleic acid of this embodiment may contain two, three, four, or five times as many florets as spikelets of a plant not containing the nucleic acid.

[0050] The method for evaluating the number of florets contained in spikelets of a plant containing the nucleic acid of this embodiment can be determined appropriately by those skilled in the art, but can be evaluated, for example, by actually dissecting and observing the plant. When observing by dissection, it is preferable to use a plant after heading, and more preferably before flowering.

[0051] This embodiment also provides a plant having the nucleic acid of this embodiment. The plant is preferably a grass plant, more preferably sorghum, but may be any other plant. The plant having the nucleic acid of this embodiment can be used for any purpose depending on the purpose. For example, the plant having the nucleic acid of this embodiment can be cultivated for food or feed.

[0052] Plants carrying the nucleic acid of this embodiment may be produced by any method known to those skilled in the art.

[0053] For example, (i) a plant mutated to have the nucleic acid of this embodiment may be selected from nature, (ii) a plant having the nucleic acid of this embodiment may be selected from artificially mutated plants, (iii) a plant having the nucleic acid of this embodiment may be obtained by crossbreeding, (iv) a plant having the nucleic acid of this embodiment may be obtained by genetic recombination or genome editing, or (v) a plant having the nucleic acid of this embodiment may be obtained by a combination of these methods. This embodiment also provides a method for producing a plant having the nucleic acid of this embodiment, and a method for producing a plant having an increased number of florets in spikelets. In (i) and (ii) above, a method for selecting a plant having the nucleic acid of this embodiment may include, for example, a selection method using the method provided as the first embodiment. In (ii) above, a method for artificially inducing mutation may include, for example, a method using radiation or a chemical substance such as ethyl methanesulfonate (EMS). As in (iii) above, a method for obtaining a plant having the nucleic acid of this embodiment by crossbreeding may include, for example, a method called DNA marker-assisted selection, which uses a DNA marker linked to the nucleic acid of this embodiment. By performing selection using such gene markers, the breeding process can be shortened, making it possible to rapidly obtain plants containing the desired gene. As described above in (iv), a method for obtaining a plant containing the nucleic acid of this embodiment by genetic recombination includes, for example, a method using Agrobacterium. When using Agrobacterium, for example, Agrobacterium containing the nucleic acid of this embodiment or a vector containing the nucleic acid is infected into a plant cell, preferably a callus, and the nucleic acid is incorporated into the plant genome in the plant cell, allowing the plant cell to redifferentiate, thereby obtaining a plant containing the nucleic acid of this embodiment. Alternatively, a plant containing the nucleic acid of this embodiment can be obtained by directly introducing the nucleic acid of this embodiment into a plant using a particle gun or the like. As described above in (iv), a method for obtaining a plant containing the nucleic acid of this embodiment by genome editing includes, for example, a method using an endonuclease such as ZFN, TALEN, or CRISPR / Cas9.When such an endonuclease is used, for example, a plant having the nucleic acid of this embodiment can be obtained by introducing the endonuclease and a gRNA that recognizes the target sequence into a plant, and then introducing the nucleic acid of this embodiment into the target site in the plant genome, or by introducing a mutation into the target site in the plant genome so that the nucleic acid of this embodiment is contained. A vector may be used to introduce the endonuclease and gRNA into the plant.

[0054] By the above method, a plant having the nucleic acid of this embodiment can be obtained. When the nucleic acid of this embodiment is introduced into a plant genome to obtain a plant having the nucleic acid of this embodiment, a plant having the nucleic acid of this embodiment on its genome can also be obtained. The plant is preferably a grass plant, more preferably sorghum, but may be any other plant.

[0055] Whether a plant contains the nucleic acid of this embodiment can be assessed by any method known to those skilled in the art. For example, DNA extracted from the plant can be amplified using any primers that amplify a region containing the nucleic acid of this embodiment, and the resulting amplification product can be analyzed to assess whether the plant contains the nucleic acid of this embodiment. Alternatively, the method provided as the first embodiment can be used to assess whether the plant contains the nucleic acid of this embodiment.

[0056] The present invention will be specifically explained below by showing examples, but the present invention is not limited to these examples.

[0057] <Phenotype Segregation Analysis of Twin Traits> A sorghum line that produces one floret per spikelet (Single parent line: SIL-05) was crossed with a line that produces two florets per spikelet (Twin parent line: IS 20679), and the F １ Furthermore, F １ F by self-fertilization of individuals ２ A group was obtained. １ The phenotype of the individuals was confirmed, and it was confirmed that the spikelets of all individuals had two florets (Figure 1). ２The phenotype of the population was confirmed, and it was found that the phenotype with two florets per spikelet (Twin) segregated to the phenotype with one floret per spikelet (Single) at a ratio of 3:1. These results indicated that the Twin parent line had one dominant gene that controlled the number of florets per spikelet.

[0058] <QTL analysis> The F obtained above ２ Seeds from 384 individuals in the population were sown in cell trays, and the seedlings grown in a greenhouse were planted in 0.6 L pots (slit pots CSM-120, Kaneya Sangyo) (1 individual per pot). Bonsol No. 1 (Sumitomo Chemical) was used as the soil, and 0.3 g each of compound fertilizer (8-8-8, Iris Ohyama Co., Ltd.) and slow-release fertilizer (LP Coat SS100, J-Cam Agri Co., Ltd.) was applied per pot. After the plants had grown to a certain size, leaves were sampled approximately 7 cm from the tip for all individuals, and DNA extracted by the CTAB method was used to create a Restriction Site Associated DNA sequence (RAD-seq) library. Using a linkage map constructed based on RAD-seq data, we performed QTL analysis using trait data from spikelets immediately prior to anthesis, evaluating them for two phenotypes: single and twin. As a result, we detected a QTL with a percent variance explained (PVE) of approximately 100% at approximately 59 Mb on chromosome 6 (Figure 2). The logarithm of odds (LOD) of this QTL was high, at 166.3. Furthermore, most individuals homozygous or heterozygous for the twin parental lineage at the nearest marker for this QTL exhibited the twin trait (Figure 3).

[0059] <Selection of responsible gene candidates> Analysis of the genomic sequence around 59 Mb on chromosome 6 confirmed that an inversion had occurred in the Twin parental line-specific region between 59.23 Mbp and 59.27 Mbp (35.614 kbp) (Figure 4). This inversion is thought to have altered the expression pattern of the WUSCHEL (WUS) gene or the AUXIN RESPONSE FACTOR 5 (ARF5) gene by acquiring a new promoter.

[0060] <Creation of Transformants (1)> Using the genomic DNA of a Twin parent line individual as a template, a region (SEQ ID NO: 1) containing WUS, ARF5, and their respective putative promoter sequences was cloned. A pBUH3-based vector incorporating this region was transformed into a transformable single-type sorghum individual (Tx430). The resulting transformant's spikelets were observed after heading and before flowering. Two florets (a floret and a revertant floret) were confirmed, demonstrating that the nucleotide sequence set forth in SEQ ID NO: 1 determines the Twin trait ( Figure 5 ). Transformation was performed by infecting callus derived from immature embryos of Tx430 with Agrobacterium (EHA105 strain) transformed with the vector (heat shock introduction). Spikelets were observed in individuals regenerated from the Agrobacterium-infected callus. The same procedure was followed below.

[0061] <Creation of Transformants (2)> To investigate whether WUS or ARF5 is the gene that determines the Twin trait, or whether simultaneous function of both genes is required for the expression of the Twin trait, vectors in which the ARF5 gene function and the WUS gene function were disrupted were prepared based on a vector into which SEQ ID NO: 1 was cloned, and single individuals were transformed in the same manner as in <Creation of Transformants (1)> above. Function was disrupted by introducing an early stop codon into the first half of each gene in the vector. As a result, two florets were observed in the transformant introduced with the vector that disrupted the ARF5 gene function, but two florets were not observed in the transformant introduced with the vector that disrupted the WUS gene function (Figure 6). These results demonstrate that WUS, which acquired a new promoter through inversion of a specific region, is the gene responsible for determining the Twin trait.

[0062] <Genotyping using PCR> PCR primers I to III were designed to identify individuals exhibiting the Twin trait at the seedling stage (SEQ ID NOS: 2 to 4, respectively). Primer I was designed to anneal to the region between the recombination site and WUS in Twin individuals and Single individuals, primer II was designed to anneal to the region between the recombination site of the complementary strand to the strand annealed by primer I and ARF5 in Twin individuals, and primer III was designed to anneal to the region between the recombination site of the complementary strand to the strand annealed by primer I and ARF5 in Single individuals. Primers I to III were all mixed together to identify the Single parent line (single parent line type homozygous), Twin parent line (twin parent line type homozygous), and F １ The results of PCR using DNA extracted from heterozygous individuals as a template are shown in Figure 7. In the Single parent line, only an amplified product (787 bp) was obtained using primers I and III, and in the Twin parent line, only an amplified product (1060 bp) was obtained using primers I and II. １ In individuals (heterozygous), these two types of amplification products were obtained, demonstrating that whether or not an individual exhibits the Twin trait can be determined before heading using primers I to III.

[0063] In the sequence shown in SEQ ID NO: 1, WUS, ARF5, the 5' regions of WUS and ARF5, and the recombination site are arranged as follows: WUS, the 5' region of WUS, the recombination site, the 5' region of ARF5, and ARF5. In Table 1, the coding region of WUS (excluding untranslated regions and including exons and introns; the same applies below) is indicated by a first underline, the recombination site is indicated by a second underline (an inversion occurs between the two underlined nucleotides), and the coding region of ARF5 is indicated by a third underline.

[0064]

Claims

1. A method for detecting that a target grass plant is a grass plant that produces two florets per spikelet, the method comprising the step of confirming the presence of a recombination site due to an inversion of a base sequence in the 5' region of the WUSCHEL gene, wherein the recombination site is adjacent to the 5' sequence of the AUXIN RESPONSE FACTOR 5 gene.

2. The method according to claim 1, wherein the inversion of the base sequence is an inversion of a double-stranded sequence containing the AUXIN RESPONSE FACTOR 5 gene.

3. The method according to claim 1 or 2, wherein the step of confirming the presence of a recombination site is a step of obtaining an amplification product containing the 5' flanking sequence and the 3' flanking sequence of the recombination site, and the length of the amplification product is 10,000 bp or less.

4. The method according to claim 3, wherein the amplification product comprises the 810th to 830th base sequence on the 5' side of the WUSCHEL gene and the 1620th to 1640th base sequence on the 5' side of the AUXIN RESPONSE FACTOR 5 gene.

5. A nucleic acid comprising a base sequence having at least 80% identity with the base sequence shown in SEQ ID NO: 1, which nucleic acid controls the number of florets per spikelet.

6. A nucleic acid consisting of the base sequence shown in SEQ ID NO:

1.

7. A vector comprising the nucleic acid of claim 5 or 6.

8. A transformant having the nucleic acid according to claim 5 or 6.

9. The transformant according to claim 8, wherein the transformant is a plant.

10. The transformant according to claim 9, wherein the plant is sorghum.

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