Mutant protein sped1-D(L) expressing high-frequency clustered spikelet trait and its coding sequence and application

Abstract

The invention discloses a mutant protein sped1-D (L) expressing a high-frequency clustered spikelet trait and its coding sequence and application. Compared with the wild-type SPED1 protein, the amino acid sequence of the mutant protein is T at the 70th residue, R at the 130th residue, L at the 414th residue, K at the 447th residue, T at the 519th residue, and the 76th amino acid residue is V or I. The full length of the SPED1 protein is 630aa, and the accession number of the full-length gene sequence encoding the wild-type SPED1 protein in Genbank is LOC_Os06g39650. The mutant protein sped1-D (L) of the invention has the function of accelerating the maturation of rice and shortening the growth period. In addition, the yield-increasing effect brought by the high-frequency clustered spikelets provides the possibility for the breeding of early-maturing and high-yield new varieties; at the same time, it is a new source of fragrance and functional nutritious rice.

Description

Technical Field

[0001] The invention relates to the technical field of plant molecular biology, and in particular to a mutant protein sped1-D (L) expressing high-frequency clustered spikelet traits, and a coding sequence and application thereof. Background Art

[0002] The number of grains per spike is one of the important factors in the yield of rice and other grass crops. The number of branches and stalks at all levels that make up the spike (inflorescence) shape and the density of spikelets on them will affect the number of grains per spike. The clustered spikelet mutation directly affects the spike shape and the number of grains per spike. Therefore, the clustered spikelet mutation not only provides a possible breeding approach to increase the number of grains per spike, but also can be directly applied to production if it is formed on excellent germplasm resources.

[0003] Clustered spikelets in rice is a mutant trait first discovered and reported by American scholar Jordan in 1957. Its phenotype is that the spikelets at the top of the main spike axis and branch stalk are attached to 1-2 spikelets below. Since then, this mutant has been discovered or created in Japan (Nagao et al., 1963) and China (Zeng Zuokui, 1999; Zhang Xianwei et al., 2013) and other places. The spikelet mutants from different sources have different spikelet morphology, fruiting rate and husk color. Some of them have well-developed floral organs, can bloom and fruit normally, have full and plump grains, and have a high fruiting rate (Tian Cui et al., 2010; Zhou Peng et al., 2012; Zhou Junjie et al., 2016); while others have low fruiting rate, smaller grain shape, darker mature husk color or deformed grains (Chen Hongqi et al., 2002; Chen Guangyi et al., 2014), especially for materials from artificial mutagenesis (Chen Hongqi et al., 2002). Classical genetic analysis shows that this trait is controlled by a single gene and is partially dominant. In terms of development, in-depth research has found that the reason for the emergence of spikelets is that the elongation of the top of the branch peduncle where the spikelet is located is inhibited during the late stage of spikelet development, when the inner and outer palea of ​​the spikelet are closed to maturity, causing its development to slow down or even stop, and then clustered together with the spikelets below. Zhang Yanli et al. (2010) conducted microscopic observation on the development of spikelets in rice double-grain mutants and found that the development of spikelets in double-grain mutants occurred during the differentiation period of the second branch primordium.

[0004] At present, many spikelet mutants from different sources have been located on chromosome 6 and are controlled by a pair of incomplete dominant genes (Nagao et al., 1963; Chen Hongqi et al., 2002; Zheng et al., 2003; Zhang Yi et al., 2006; Xiao Qiaozhen, 2007) or a pair of recessive spikelet genes (Zhou Peng et al., 2012; Zhang Yanli et al., 2010). On chromosome 4, Zeng Xiuhong (2009) and Meng Lang (2011) each located a recessive mutant spikelet gene, cl-4 and cll(t), and found that their mutation sites were both located on the same cytochrome P450 90B1 gene. cl-4 is caused by a single base T deletion on exon 1, while cll(t) is caused by a single base GA substitution on exon 4. In 2014, Jiang et al. cloned the rice clustered spikelet gene located on chromosome 6 from a mutant of rice 9311. The gene encodes a triangular pentapeptide repeat protein. The phenotype after mutation is partially dominant short pedicels (shortened pedicels and or second branches 1), so the gene was named sped1-D. The two detected SNP mutation sites occurred at amino acids 70 and 414. In addition to shortening the pedicels and secondary branches and clustering the spikelets, the spedl-D mutation also affects the fruit set rate. With the increase of clustering, the fruit set rate and 1000-grain weight also decrease. Transcriptome analysis found that the expression of GID1L2 family members was downregulated, indicating that the gibberellin signaling pathway is involved in the elongation of pedicels and secondary branches (Jiang et al., 2014). In the same year, Guo et al. (2014) cloned the clustered spikelet gene located on chromosome 4. Its translation product is a cytochrome P450 724B1 protein, which is involved in the brassinolide biosynthesis pathway and affects spikelet clustering and plant height formation. The mutant is due to the deletion of the T in the first exon, which leads to premature termination of translation. Chen Guangyi et al. (2014) also located the same clustered spikelet gene from this site, but found that its mutation site is located on the fourth exon, which is a single base C 1810 Replace with T 1810 mutation. In 2018, Zheng Yanmei et al. located a new sped1-D allele mutant on chromosome 6, named OsCl-6, but the gene was later cloned by Long Tuan et al. (2020) and named sped1-1 (authorized patent number is ZL202010658850.6). In 2024, Zhang Xiaoxing et al. located and cloned a new BRD3 gene (LOC_Os06g39880) encoding a BR metabolic enzyme on chromosome 6, and discovered for the first time that BR has the function of determining the number of grains per panicle by regulating the secondary branching of rice panicles. In compound-grain rice, BRD3 is only moderately activated by structural variations in the CL locus in specific tissues, thereby reducing the BR content, leading to the formation of clustered spikelets.

[0005] Currently, there is an urgent need in the art for new rice high-frequency clustered spikelet mutants and detection methods and applications thereof for improving the number of grains per spike and breeding high-yield rice varieties. Summary of the invention

[0006] The present invention has discovered a mutant protein sped1-D (L) that determines the high-frequency clustered spikelet trait of rice through research. It is partially dominant and can make rice develop into a spikelet consisting of 2-12 or even more grains with a frequency of up to about 70% (±25%), increase the number of grains per spike, accelerate maturity, and shorten the growth period to 95 days. The produced rice produces and accumulates a variety of aroma components and functional nutrients that are different from the classic aroma substance 2-acetyl-1-pyrroline (2-AP), making the rice fragrant and rich in functional nutrients. Therefore, the primary purpose of the present invention is to provide a mutant protein sped1-D (L) that expresses the high-frequency clustered spikelet trait and its coding sequence and application. The second purpose of the present invention is to provide a high-frequency clustered spikelet and a method for cultivating the same. The third purpose of the present invention is to provide a method for identifying rice with the high-frequency clustered spikelet trait. The fourth purpose of the present invention is to provide a paddy (rice) product.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] As the first aspect of the present invention, a mutant protein sped1-D (L) expressing a high-frequency clustered spikelet trait is provided. Compared with the wild-type SPED1 protein, the amino acid sequence of the mutant protein is T at residue 70, R at residue 130, L at residue 414, K at residue 447, T at residue 519, and the amino acid residue at position 76 is V or I. The full length of the SPED1 protein is 630 aa, and the accession number of the full-length gene sequence encoding the wild-type SPED1 protein in Genbank is LOC_Os06g39650.

[0009] According to the present invention, the mutant protein sped1-D(L) is selected from the proteins described in any one of the following items (1) to (3):

[0010] (1) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1;

[0011] (2) a protein having the activity of determining the high-frequency clustered spikelet trait of rice as described in (1) by substituting and / or deleting and / or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO: 1; and

[0012] (3) A protein derived from rice that has at least 90%, preferably at least 95%, and more preferably at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 1 and has the activity of determining the high-frequency clustered spikelet trait of rice as described in (1).

[0013] According to the present invention, the mutant protein sped1-D(L) exists in a monomeric form.

[0014] As the second aspect of the present invention, an isolated polynucleotide encodes the mutant protein sped1-D(L) described above.

[0015] According to the present invention, the polynucleotide is selected from the polynucleotide described in any one of the following items (1) to (4):

[0016] (1) a polynucleotide consisting of the sequence shown in SEQ ID NO: 2;

[0017] (2) a polynucleotide that differs from the sequence of SEQ ID NO: 2 only due to the degeneracy of the nucleotide code and encodes the mutant protein sped1-D(L) as described above;

[0018] (3) a polynucleotide that hybridizes with the complementary sequence of the sequence shown in SEQ ID NO: 2 under stringent conditions and encodes the mutant protein sped1-D(L) described above; and

[0019] (4) A polynucleotide derived from rice that has at least 90%, preferably at least 95%, and more preferably at least 99% sequence identity with the sequence shown in SEQ ID NO: 2 and encodes the mutant protein sped1-D(L) described above.

[0020] As the third aspect of the present invention, a method for breeding clustered spikelet rice comprises introducing the above-mentioned polynucleotide into the target rice genome, and breeding heterozygous or homozygous genotypes expressing high-frequency clustered spikelet traits in its offspring, thereby obtaining clustered spikelet rice.

[0021] According to the present invention, the method for breeding clustered spikelet rice is to introduce the polynucleotide into the target rice genome through hybrid breeding, backcross breeding or gene editing technology, assisted by the selection of SNP markers at positions 226, 388 and 1341 in the sequence shown in SEQ ID NO:2 or artificially developed CAPS markers, so as to breed the clustered spikelet rice.

[0022] Furthermore, the CAPS marker includes using the SNP site (C / A single nucleotide polymorphism) located at position 368 in the sequence shown in SEQ ID NO: 3 as a target site.

[0023] Furthermore, the CAPS marker-assisted selection comprises the following steps:

[0024] a) performing PCR amplification on a sample from a rice plant to be bred using the primers shown in SEQ ID NO: 4 and SEQ ID NO: 5;

[0025] b1) using a first restriction endonuclease to restrictively cut the amplified product of step a), separating and detecting the length of the resulting cut product, wherein the first restriction endonuclease is selected from Asp26HI, Asp27HI, Asp35HI, Asp36HI, Asp40HI, Asp50HI, BsaMI, BscCI, BsmI, Uba1382I, Uba1415I, or Mva1269I; or

[0026] b2) using a second restriction endonuclease to restrictively cut the amplified product of step a), separating and detecting the length of the resulting cut product, wherein the second restriction endonuclease is selected from BbsI, Bbv16II, BbvII, Bco102II, BsaVI, Bsc91I, BspBS31I, BspIS4I, BspVI, BstBS32I, BstTS5I, BsvTS514I, BpiI, or BpuAI; and

[0027] c) breeding rice plants in which the SNP site is a C / A base.

[0028] As the fourth aspect of the present invention, a method for identifying rice with a high-frequency clustered spikelet trait comprises detecting the polynucleotide as described above in a sample from the rice, wherein if the presence of a C / A base at the SNP site is detected, it indicates that the rice has a high-frequency clustered spikelet trait.

[0029] As the fifth aspect of the present invention, a rice product is made from rice containing the mutant protein sped1-D(L) described above, rice containing the polynucleotide described above, high-frequency clustered spikelet rice bred by the clustered spikelet rice breeding method described above, or rice identified by the method described above, or contains ingredients derived from the rice, wherein the rice product has been processed and cannot be used as rice breeding material.

[0030] According to the present invention, the rice product is selected from the group including but not limited to brown rice, polished rice, cooked rice, canned rice, a-cooked rice, self-heating rice, rice flour, rice noodles, rice cakes, rice balls and rice milk beverages.

[0031] According to the present invention, the rice product, wherein the rice product has a flavor profile that does not contain 2-acetyl-1-pyrroline, preferably, the flavor profile comprises limonene, anethole, methyl myristate, galaxol, trimethyl borate, nonanal and benzaldehyde, more preferably, the flavor profile comprises limonene, anethole, methyl myristate, galaxol, trimethyl borate, nonanal, benzyl benzoate, mannitol and 2-(decanoyloxy)propane-1,3-diyl dioctanoate.

[0032] As a sixth aspect of the present invention, the mutant protein sped1-D(L) or the polynucleotide described above is used in rice breeding or germplasm resource improvement.

[0033] According to the present invention, in the above-mentioned use, compared with a reference rice without the mutant protein sped1-D(L) or the polynucleotide, the rice expressing the mutant protein sped1-D(L) or the polynucleotide has a trait selected from the following:

[0034] clustered spikelets with increasing frequency of occurrence;

[0035] shortened spikelet stalk length;

[0036] increased kernel number per ear and yield;

[0037] shortened reproductive period; and / or,

[0038] The flavor profile does not contain 2-acetyl-1-pyrroline (2-AP).

[0039] Beneficial effects of the present invention:

[0040] (1) The present invention provides a new mutant protein sped1-D(L) that expresses high-frequency clustered spikelet traits and its molecular marker and detection method, which provides new germplasm resources and approaches for improving the number of grains per spike and breeding high-yield rice varieties.

[0041] (2) The mutant protein sped1-D(L) expressing the high-frequency clustered spikelet trait provided by the present invention has the function of accelerating the maturity of rice and shortening the growth period. In addition, the yield-increasing effect brought about by the high-frequency clustered spikelets provides a possibility for the breeding of new early-maturing and high-yielding varieties.

[0042] (3) The mutant sped1-D(L) expressing the high-frequency clustered spikelet trait provided by the present invention also has the function of enabling rice to produce and accumulate a variety of aroma components and functional nutrients that are different from the classic aroma component 2-acetyl-1-pyrroline, and is a new source of aroma and functional nutrition for rice. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1: Comparison of the spikelet mutant and its derivatives with the male parent Lemont. In the figure, ①. The spikelet mutant spikelet type; ②. The spikelet type of the new spikelet line derived from the spikelet mutant, Linlongxin No. 1; ③. The spikelet type of the new spikelet line derived from the spikelet mutant, Linlongxin No. 2; ④. The spike type of the male parent of the new spikelet line, Lemont.

[0044] Figure 2 :Analysis of the difference in mutation sites between the rice clustered spikelet mutant sped1-D(L) created by Linlong Biotechnology in the present invention and the publicly reported rice clustered spikelet mutants sped1-D (Jiang et al., 2014) and sped1-1 (Long Tuan et al., 2020). A, the comparison of the nucleic acid coding sequences of the clustered spikelet mutant sped1-D(L) and the wild-type SPED1, the single nucleotide polymorphism sites that cause amino acid substitution missense mutations are marked in red, and only the segments of the aligned sequences with polymorphic sites are shown; B, the correspondence of the mutation sites of the clustered spikelet mutant sped1-D(L) created by Linlong Biotechnology at the protein level with the wild-type SPED1 and the similarities and differences between the replaced amino acid residues and the other two allelic mutants sped1-D and sped1-1. The full length of the wild-type SPED1 protein is 630aa, and the accession number of the full-length gene sequence encoding the protein in Genbank is LOC_Os06g39650.

[0045] Figure 3 : The three-dimensional structures of sped1(1), sped1-D(L)(2), sped1-D(3) and sped1-D(L) predicted by SWISS model after the 76th mutant amino acid residue I was changed back to the wild-type amino acid residue V(4). The different parts of the three-dimensional structures of 1 and 3 and 2 and 4 are shown with black arrows.

[0046] Figure 4 : The application of the artificially developed CAPS marker in the hybrid breeding program of this embodiment. DETAILED DESCRIPTION

[0047] In order to enable those skilled in the art to more fully understand the technical solution of the present invention, exemplary embodiments of the present invention will be described in more comprehensive and detailed manner below in conjunction with the accompanying drawings.

[0048] 1. Product Source

[0049] (1) sped1-D(L) of the present invention

[0050] A mutant protein that determines the high-frequency clustered spikelet trait, which exists in monomeric form and is different from the wild non-clustered spikelet SPED1 protein, which exists in the plant as a dimer. The sped1-D(L) mutant protein is also different from the sped1-D and sped1-1 proteins that form 2-3 spikelets at the top of the branch caused by the SPED1 mutation, which exist in the plant as deformed dimers. Different spatial structures lead to different functional effects of the proteins.

[0051] (2) sped1-D (for details, see reference Jiang et al-Regulation of Inflorescence Branch Development in rice through a novel pathway involving the pentatricopeptiderepeat protein sped1-D-2014).

[0052] (3) sped1-1 (authorized patent number is ZL202010658850.6).

[0053] (4) The full length of the wild-type SPED1 protein is 630 aa, and the accession number of the full-length gene sequence encoding the protein in Genbank is LOC_Os06g39650. The protein sequence of SPED1 is shown in SEQ ID NO: 10. The DNA sequence of SPED1 is shown in SEQ ID NO: 11.

[0054] 2. Terms related to amino acid sequences and polynucleotide sequences

[0055] As used herein, "stringent conditions" encompass conditions under which hybridization occurs only when the mismatch between the hybridizing molecule and the DNA target is less than 50%. "Stringent conditions" include further stringent specific levels. Thus, as used herein, "medium stringency" conditions refer to conditions under which molecules with a sequence mismatch rate of more than 50% will not hybridize; "high stringency" conditions refer to conditions under which sequences with a mismatch rate of more than 20% will not hybridize; and "very high stringency" conditions refer to conditions under which sequences with a mismatch rate of more than 10% will not hybridize.

[0056] In a specific embodiment, stringent conditions may include hybridization at 65°C, followed by a wash in 0.1 x SSC / 0.1% SDS at 65°C for 40 minutes.

[0057] The following are representative, non-limiting hybridization conditions:

[0058] Very high stringency: hybridization in 5x SSC buffer at 65°C for 16 hours; wash twice in 2x SSC buffer at room temperature for 15 minutes each; and wash twice in 0.5x SSC buffer at 65°C for 20 minutes each.

[0059] High stringency: hybridization in 5x-6x SSC buffer at 65°C-70°C for 16-20 hours; wash twice in 2xSSC buffer at room temperature for 5-20 minutes each; and wash twice in 1x SSC buffer at 55°C-70°C for 30 minutes each.

[0060] Medium stringency: hybridize in 6x SSC buffer at room temperature to 55°C for 16-20 hours; wash at least twice in 2x-3x SSC buffer at room temperature to 55°C for 20-30 minutes each.

[0061] In a specific embodiment, the nucleic acid molecules of specific hybridization can keep combining under very high stringency hybridization conditions. In these and further embodiments, the nucleic acid molecules of specific hybridization can keep combining under high stringency hybridization conditions. In these and further embodiments, the nucleic acid molecules of specific hybridization can keep combining under medium stringency hybridization conditions.

[0062] As used herein, the term "oligonucleotide" refers to a short nucleic acid polymer. Oligonucleotides can be formed by cutting longer nucleic acid segments or by polymerizing single nucleotide precursors. Automated synthesizers allow the synthesis of oligonucleotides up to hundreds of base pairs in length. Because oligonucleotides can be combined with complementary nucleotide sequences, they can be used as probes for detecting DNA or RNA. The oligonucleotides (oligodeoxyribonucleotides) composed of DNA can be used for PCR (technology for amplifying small DNA sequences). In PCR, oligonucleotides are typically referred to as "primers", which allow DNA polymerase to extend oligonucleotides and replicate complementary chains.

[0063] The terms "percent sequence identity" or "percent identity" or "identity" are used interchangeably and refer to a sequence comparison based on identical matches between corresponding identical positions in the sequences being compared between two or more amino acid or nucleotide sequences. Percent identity refers to the degree to which two optimally aligned polynucleotide or peptide sequences are invariant over the comparison window of components, such as nucleotides or amino acids. Hybridization experiments and mathematical algorithms known in the art can be used to determine percent identity. There are many mathematical algorithms as sequence alignment computer programs known in the art that calculate percent identity of sequences. These programs can be divided into global sequence alignment programs or local sequence alignment programs.

[0064] The term "similarity" refers to a comparison between amino acid sequences, and not only the identical amino acids at corresponding positions are considered, but also the functionally similar amino acids at corresponding positions are considered. Therefore, in addition to sequence similarity, the similarity between polypeptide sequences also indicates functional similarity.

[0065] The term "homology" is sometimes used to refer to the level of similarity between two or more nucleic acid or amino acid sequences, expressed as a percentage of positional identity (i.e., sequence similarity or identity). Homology also refers to the concept of evolutionary relatedness, often demonstrated by similar functional properties between different nucleic acids or proteins that share similar sequences.

[0066] As used herein, the term "variant" refers to substantially similar sequences. For nucleotide sequences, naturally occurring variants can be identified using molecular biology techniques well known in the art, such as polymerase chain reaction (PCR) and hybridization techniques as outlined herein.

[0067] For nucleotide sequences, variants are included in the deletion and / or addition of one or more nucleotides at one or more internal sites in natural polynucleotides, and / or the replacement of one or more nucleotides at one or more sites in natural polynucleotides. As used herein, "natural" nucleotide sequences include naturally occurring nucleotide sequences. With respect to nucleotide sequences, molecular biology techniques well known in the art can be used to identify naturally occurring variants, for example, using the polymerase chain reaction (PCR) and hybridization techniques outlined below to identify. Nucleotide sequence variants also include nucleotide sequences of synthetic origin, such as those produced using site-directed mutagenesis techniques. Typically, as determined by the sequence alignment programs and parameters described elsewhere herein, the variants of the specific nucleotide sequences of the present invention will have at least about 40%, 45%, 50%>, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the specific nucleotide sequence. Biologically active variants of the nucleotide sequences of the present invention may differ from the sequence by only 1 to 15, only 1 to 10, such as 6 to 10, only 5, only 4, 3, 2, or even only 1 nucleic acid residue.

[0068] As used herein, the term "operably linked" refers to a first nucleic acid sequence being operably linked to a second nucleic acid sequence when the first nucleic acid sequence has a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence when it affects the transcription or expression of the coding sequence. When produced recombinantly, operably linked nucleic acid sequences are generally contiguous and, in the case of two protein coding regions that need to be linked, are in the same reading frame. However, elements do not have to be contiguous to be operably linked.

[0069] 3. High-frequency clustered spikelet rice of the present invention

[0070] The invention relates to a rice plant that inherits the sped1-D (L) mutant protein coding sequence, and is characterized in that: the rice plant develops spikelets with a frequency of about 70% (±25%) consisting of 2-12 or even more grains; the rice plant matures quickly, and the growth period can be as short as 95 days; the rice grains produce and accumulate a variety of flavor components and functional nutrients that are different from the classic flavor substance 2-acetyl-1-pyrroline (2-AP), making the rice fragrant and nutritious.

[0071] 4. CAPS marker-assisted selection

[0072] CAPS marker-assisted selection is a type of marker-assisted selection (MAS). Molecular marker-assisted selection is a new technology that has emerged with the rapid development of modern molecular biology technology. It can use a variety of different molecular markers to quickly and accurately analyze the genetic composition of individuals at the molecular level, thereby achieving direct selection of genotypes and conducting molecular breeding. Commonly used molecular markers include but are not limited to simple sequence repeat markers (SSR, Simple Sequence Repeats), single nucleotide polymorphism markers (SNP, Single Nucleotide Polymorphism), restriction fragment length polymorphism markers (RFLP, Restriction Fragment length Polymorphism) and enzyme-amplified polymorphic sequence markers (CAPS, Cleaved Amplified Polymorphic Sequences), etc.

[0073] 5. Paddy (rice) products and their processing

[0074] Paddy rice products are various products formed by artificial processing of paddy rice, including brown rice, which is the caryopsis after the outer protective cortical husk of rice is removed, and its endosperm commonly known as the bran layer and the embryo are almost completely retained; polished rice, which is the polished white rice after the bran layer and embryo are removed after fine grinding; cooked rice, which is made by washing and steaming with water; canned rice, which is made by placing an appropriate amount of rice and water in a metal can, steaming, curling, and heating to sterilize; a-cured rice, which is made by washing and soaking rice, steaming or boiling it, and then drying it with hot air; self-heating rice, which comes with a heating pack, and heats up within 3-5 seconds after meeting water, and the temperature reaches above 150°C, the steam temperature reaches 200°C, and the longest insulation time can be up to 3 hours, so it is easy to cook raw rice into cooked rice. Rice noodles are noodles made from rice. The famous ones include Fujian's "Xinghua noodles", Guangdong's "Shahe noodles", Yunnan's "Guoqiao rice noodles" and Sichuan's "Yuechi rice noodles". The processing process includes washing rice, soaking → grinding → steaming powder → pressing sheets, squeezing threads → re-steaming → cooling → drying → eating rice noodles; rice cakes are steamed with sticky rice or rice flour; rice balls are made by washing, drying, grinding rice into broken rice flour, steaming it in a pot, adding appropriate amount of boiling water and salt to make it into balls, and then adding stuffing to knead it into balls and steaming it; rice milk beverage is made by washing, soaking, grinding and homogenizing brown rice or polished rice, filtering and heating to sterilize it.

[0075] 6. Flavor spectrum

[0076] The so-called flavor spectrum is the sum of volatile aroma substances produced by rice or its cooked rice. The volatile aroma substances of rice are mainly formed by carbohydrates (terpenes, furanones and pyrones), fatty acids [aldehydes and alcohols generated by lipoxygenase (LOX) or α- and β-oxidase] and amino acids (acids, alcohols, aldehydes, esters, lactones, and flavor molecules containing N and S, benzene and phenylpropane volatile compounds). Carbonyl compounds can produce native and strong aromas, while volatile alcohols produce softer odors. The source of ketone compounds is mainly the oxidation or thermal degradation of polyunsaturated fatty acids, amino acid degradation or microbial oxidation. They usually have a special aroma, most of which are fresh aromas (floral and fruity), and the aroma is excellent and long-lasting. The flavor spectrum of rice from different varieties is different. The classic aroma substance in rice is 2-acetyl-1-pyrroline (2-AP).

[0077] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0078] Example 1: Obtaining and genetic analysis of the rice high-frequency clustered spikelet mutant sped1-D(L)

[0079] In August 2020, the inventors unexpectedly obtained a mutant from the chemically induced seeds of Shuangdwarf 85, with 2-7 spikelets clustered together and a frequency of about 75% of the whole spikelet ( Figure 1 -①), then cut the stump and regenerate it, and divide the stump three times to form 8 stumps of asexual reproduction, of which 4 stumps are allowed to grow to maturity for harvesting, and the other 4 stumps are cut and regenerated for use with the male parents (lemont, GUNONG 13, and HT54 are used in this embodiment) with a maturity period similar to that in the original material garden and cut at the same time, and warm soup emasculation and hybridization are carried out to obtain F1, and after the hybrids are enriched for 18-22 days, they are immediately harvested and turned over for autumn planting and greenhouse cultivation (rented). It was found that all hybrid F1 showed clusters of 2 spikelets at the end of the branch stalk, and the growth period tended to be shorter, indicating that the clustered spikelet mutant is heritable, the phenotype is partially dominant and ear direct (the phenotype can be directly observed in the ear), and the growth period can be shortened. In December of that year, F2 populations of different hybrid combinations were sown in southern breeding. It was found that each population was able to separate various phenotypes, including the wild type and clusters of 2, 3, 4, 5 and more spikelets at the end of the branch. The segregation ratios of clustered spikelet individuals and wild type individuals were statistically consistent with the 3:1 segregation ratio of Mendelian inheritance by the chi-square test (Table 1).

[0080] Table 1 Statistics and analysis of the genetic ratio of clustered spikelets in F2 populations of hybrids between mutants and different male parents

[0081]

[0082] Subsequently, the individual plants were harvested and planted into families according to the number of clustered spikelets, and their offspring were continuously selected by the clustered spikelet phenotype of the spikelet as a marker, until a few homozygous and stable mutant pure lines with 3-7 spikelets clustered together and a frequency of about 70% were obtained in the F5 generation during the southern breeding period of 2021-2022, and were named Linlongxin No. 1, No. 2 and No. 3 ( Figure 1 -② and Figure 1 -③, Linlongxin No. 3 is not shown) for subsequent molecular testing and phenotypic verification.

[0083] Example 2: Homologous amplification and allelic sequencing analysis of the rice high-frequency clustered spikelet mutant sped1-D(L)

[0084] Following Example 1, the inventors used the nucleic acid coding sequence and protein polypeptide sequence of the clustered spikelet mutant sped1-D cloned by Jiang et al. in 2014 as reference sequences to perform a PCR reaction on the Linlongxin 1 ( Figure 1 -②) and its clustered spikelet mutant ( Figure 1 -①)Maternal parent and Lemont( Figure 1 -④) Homologous amplification and allelic sequencing analysis were performed on the male parent, and the analysis was repeated three times per plant.

[0085] In this embodiment, the template DNA used for homologous amplification was extracted by CTAB method, and its concentration was measured by Nanorop2000, and it was used when diluted to 10ng / L. The primers used for homologous amplification were designed according to the reference sequence, and their nucleic acid sequences and amplification product lengths are listed in Table 2. The PCR reaction system used for homologous amplification is: 1μL 10× reaction buffer, 0.25μL dNTP, 0.25μL forward primer and 0.25μL reverse primer, 0.5U high-fidelity Taq enzyme, 1μL 10ng / μL template DNA, and ultrapure water was added to make the total volume 10μL. The PCR reaction program used for homologous amplification is: 94-98℃ denaturation for 1-3min, and then perform the following cycle: 95℃ denaturation for 20s, 57-59℃ renaturation for 20s, 72℃ extension for 60-90s, 28-32 cycles. After the cycle is completed, 72℃ is supplemented for extension for 3-10min to terminate the reaction. After the PCR amplification is completed, 0.8% agarose gel is prepared and electrophoresis is performed at 5 V / cm electric field for 40-50 min; the PCR product is recovered using a DNA gel recovery kit.

[0086] Table 2 Names and sequences of primer pairs used for homologous amplification based on sped1-D reference sequence

[0087]

[0088]

[0089] The recovered DNA of Linlongxin No. 1 and its clustered spikelet mutant maternal parent and Lemont paternal parent were sent to the Guangzhou Sequencing Department of Bioengineering for sequencing. The results showed that six single-base substitution missense mutations that caused changes in the encoded amino acid residues were detected in the pure line of Linlongxin No. 1 and its original clustered spikelet mutant within the coding sequence shown in the reference sequence ( Figure 2 A), and the two point mutation sites, position 70 and position 414, including the replaced amino acid residues are the same as sped1-D, and the other mutation site, position 519, is the same as the mutation site of sped1-1, but the replaced amino acid residue is different ( Figure 2 B) sped1-1 and sped1-D both have mutations in the SPED1 gene, but the sites of the point mutations are different ( Figure 2 B). These results confirm that the clustered spikelet mutant created by the present invention is also allelic to sped1-D and sped1-1, but has 4 and 5 more point mutation sites than them, respectively, indicating that it is a completely new mutant and is named sped1-D(L), where L represents the mutant created by Linlong Biotechnology Development (Shanghai) Co., Ltd. (referred to as Linlong Biotechnology).

[0090] Example 3: Protein spatial structure prediction and differential analysis of rice spikelet mutants sped1-D(L), sped1-D and sped1-1

[0091] The spatial structure of a protein determines its biological function. Reported data show that the reason why sped1-D forms clustered spikelets is that the three-dimensional structural changes of the protein after mutation cause the downregulation of the expression of multiple GID1L2 gibberellin receptor family cluster members involved in the elongation of spikelet stalks and secondary branch stalks (Jiang et al., 2014).

[0092] In this example, the SWISS model (https: / / swissmodel.expasy.org / interactive / XXLutc / models / ) and the nucleic acid sequence measured in Example 2 were used to predict and analyze the three-dimensional structure of the sped1-D(L) protein to analyze why the frequency of clustered spikelets caused by sped1-D(L) is higher than that of the other two mutants sped1-D and sped1-1.

[0093] The results showed that the six missense mutations of sped1-D(L) caused the protein to change from a wild-type dimer to a Figure 3-1 ) becomes a monomer ( Figure 3-2 ), while sped1-D and sped1-1 also have changes in three-dimensional structure before and after mutation, but they still maintain the wild-type dimer form, forming a deformed dimer ( Figure 3-3 , the arrows indicate the structural changes, and the three-dimensional structure of sped1-1 is not shown in the figure). However, when the inventors reduced the number of missense mutations of sped1-D(L) from 6 to 5 to 3 in the order of mutation sites, a total of 18 ways. The results showed that among the 6 mutant residues, only when the mutant residue I at position 76 was changed back to the wild type V did the monomer form of the sped1-D(L) mutant not change ( Figure 3-4 ), although the local spatial configuration of its three-dimensional structure has changed ( Figure 3-2 and 3-4 arrows); the other methods all convert it back to a dimer, but the location and degree of the three-dimensional structure change are different ( Figure 3This result shows that whether the amino acid residue at position 76 is the wild type V or the mutant I, it does not affect the existence of the mutant protein as a monomer, and the T residue at position 70, the R residue at position 130, the L residue at position 414, the K residue at position 447 and the T residue at position 519 must exist at the same time to cause the protein to change from the dimer of SPED1 to the monomer form of sped1-D (L). In other words, the amino acid residues corresponding to the above five sites in SPED1 are mutated in the manner of sped1-D (L) at the same time, which is a necessary and sufficient condition for the formation of a monomer. This is an important discovery after the clustered spikelet mutant of the present invention was created, and it is also the key to distinguish the clustered spikelet mutant of the present invention from other allelic mutants sped1-D and sped1-1.

[0094] Example 4: Molecular marker design and co-segregation identification of sped1-D(L) mutation site

[0095] Considering that the sped1-D(L) mutant has more mutation sites, which may cause the difference in restriction recognition site sequence, the inventors first analyzed the restriction enzyme cleavage site of sped1-D(L) and its wild-type SPED1 nucleic acid sequence according to the sequencing results of Example 2. The results showed that sped1-D(L) has a polymorphic C / A site at position 1556 relative to the wild-type nucleic acid sequence ( Figure 2 There are differences in the recognition of restriction endonucleases at the position A, among which 12 isozymes recognize the A base at this position, which are: Asp26HI, Asp27HI, Asp35HI, Asp36HI, Asp40HI, Asp50HI, BsaMI, BscCI, BsmI, Uba1382I, Uba1415I and Mva1269I; and 14 isozymes recognize the C base at this position, which are: BbsI, Bbv16II, BbvII, Bco102II, BsaVI, Bsc91I, BspBS31I, BspIS4I, BspVI, BstBS32I, BstTS5I, BsvTS514I, BpiI and BpuAI. Therefore, this position can be used to develop CAPS markers.

[0096] Next, according to the sequencing results of Example 2, the inventors designed gene-specific primers around the polymorphic C / A site, including a forward primer SDL_F composed of the nucleotide sequence shown in Sequence 4 and a reverse primer SDL_R composed of the nucleotide sequence shown in Sequence 5. The product sequence amplified by this pair of primers is shown in Sequence 3, with a length of 740 bp, and the polymorphic C / A site contained therein is located at the 368th base of the sequence.

[0097] Next, the inventors used the gene-specific primers SDL_F and SDL_R designed in the above steps to perform PCR amplification on Linlongxin No. 1, No. 2 and No. 3 with high-frequency clustered spikelet traits, their parents and three spikelet-top clustered plants (i.e., the high-generation (F4-F6) materials of the original mutant and Lemont hybrid in Example 1). The template DNA used was prepared according to the method and steps described in Example 2, and the reaction system used for PCR amplification was: 1 μL 10× reaction buffer, 0.25 μL dNTP, 0.25 μL forward primer and 0.25 μL reverse primer, 0.5 U Taq enzyme, 1 μL 10 ng / μL template DNA, and ultrapure water was added to make the total volume 10 μL. The reaction program used was: 94-98°C denaturation for 1-3 min, and then the following cycles were performed: 95°C denaturation for 20 s, 57-59°C renaturation for 20 s, 72°C extension for 30 s, and 32-35 cycles. After the cycle is completed, the reaction is terminated by additional extension at 72°C for 3-10 minutes. After the PCR amplification is completed, a 1% agarose gel is prepared and electrophoresed at 5V / cm electric field for 40-50 minutes; the PCR product is recovered using a DNA gel recovery kit.

[0098] Next, the inventors used a restriction endonuclease BsmI (Mva1269I) (Cat. No. ER0962, Thermo Fisher Scientific) that can recognize the recognition sequence with A base at the polymorphic site to treat the PCR product recovered in the above step for 2 hours or overnight. After the treatment, 1 μl of 10X DNA loading buffer was added to terminate the enzyme digestion reaction and loading.

[0099] The PCR products treated with restriction endonucleases were separated and detected by 1% agarose gel electrophoresis. Agarose gel preparation and electrophoresis steps: 1) 1 g agarose (Cat. No. A620014-0100 100G, Shanghai Bioengineering (Shanghai) Co., Ltd.) was dissolved in 100 ml TAE (Cat. No. B040123-0010 , Shanghai Bioengineering (Shanghai) Co., Ltd.); 2) add 7μl Gillgreen non-toxic nucleic acid dye (Cat. No. 0419-1, Beijing Huayueyang Biotechnology Co., Ltd.), pour into the agarose electrophoresis gel plate, insert the comb, spot the sample after solidification, and add the PCR product to the spotting well; 3) electrophoresis for about 30 minutes at 120V. After the electrophoresis is completed, observe the PCR product bands in the gel imaging analysis system (Model: GelDOC XR+ / 60, Bio-Rad Biomedical Products (Shanghai) Co., Ltd.) and take pictures.

[0100] The test results showed that the selected lines of Linlongxin 1, 2 and 3 with high-frequency clustered spikelets showed a 740bp band that was not cut by restriction endonuclease, just like their paternal parent, the clustered spikelet mutant. Their paternal parent, Lemont, showed only two bands of about 370bp that were cut by restriction endonuclease, which were similar in size, because it was a non-clustered spikelet. The three isolated plants with two spikelets clustered at the top of the main spike and branch showed two bands of 740bp and 370bp at the same time. Figure 4 ).

[0101] The results of this example show that the CAPS marker designed based on the C / A polymorphic site 1556 between the clustered spikelet mutant sped1-D(L) and the wild-type SPED1, including the PCR amplification primers on both sides and the restriction endonuclease that recognizes and cuts the site, is effective and accurate for co-segregation identification and seedling selection of sped1-D(L) in hybrid breeding programs.

[0102] Example 5: Identification of agronomic traits of Linlongxin 1, 2 and 3 derived from high-frequency clustered spikelet mutants

[0103] In this example, the inventors conducted a field variety yield comparison test (referred to as variety comparison test) for two consecutive seasons (after wheat and rice stubble and National Day rice stubble unique to Shanghai) on the agronomic traits of the high-frequency clustered spikelets mutant derivative lines Linlongxin No. 1, No. 2 and No. 3 obtained in Example 1. The purpose was to observe whether the sped1-D (L) mutant created by the present invention leads to high-frequency clustering of spikelets throughout the ear, and whether its number of grains per ear and yield will be increased as a result?

[0104] The control variety used in the post-wheat rice crop is Yinxiang 38, and the control variety used in the National Day rice crop is Songzaoxiang No. 1 (a geographical indication variety in Songjiang District, Shanghai). The field variety comparison test adopts a randomized block design with 3 replicates. The plot area is 15.33M 2 , seedlings were transplanted manually, the planting size was 19.8CM X 19.8CM, and the walkway width was 50CM. Pesticides were only used to prevent insects during the entire growth period, but not to prevent diseases. Fertilizer and water management was carried out according to the implementation standards of the control varieties, the amount of N fertilizer used was 25 standard N, and the ratio of N:P:K was 1:0.4:0.6. The materials for the post-wheat rice variety comparison were sown on May 25 and transplanted on June 14; the materials for the National Day rice variety comparison were sown on June 15 and transplanted on July 5. In both groups of variety comparison experiments, a repeated stump (3 stumps) near the edge of the field was selected at the seedling stage to record the tillering dynamics.

[0105] In addition to the variety comparison test, a direct seeding yield comparison experiment was also conducted on Linlongxin No. 1, and the National Day rice control Songzaoxiang No. 1 and the wheat-after rice control Yinxiang 38 were also used as references. All test materials were sown on June 15, with a sowing rate of 6 jin / mu. The sowing area was 0.8 mu for Linlongxin No. 1, 0.4 mu for Songzaoxiang No. 1 and Yinxiang 38 respectively. The width of the direct seeding opening was 3 meters and the furrow width was 0.4 meters. Other water, fertilizer, disease and pest management were the same as the variety comparison test.

[0106] Table 3 Growth performance of Linlongxin 1, 2 and 3 in two consecutive seasons of strain comparison trials (2023, Chongming, Shanghai)

[0107]

[0108] The agronomic trait observation, seed test data and yield comparison results of the strain comparison test of this embodiment are listed in Tables 3-5 respectively. From the agronomic trait observation and seed test data in Table 3, it can be seen that the Linlongxin No. 1, No. 2 and No. 3 derived from the high-frequency clustered spikelet mutant sped1-D (L) are cultivated as wheat-after-rice stubble, and their sowing start period is 23 days, 17 days and 16 days earlier than the control Yinxiang 38, and 13 days, 7 days and 6 days earlier than Songzaoxiang No. 1, respectively; when cultivated as the Guoqing rice stubble, they are 17 days, 9 days and 13 days earlier than the control Yinxiang 38, and 8 days, 0 days and 4 days earlier than Songzaoxiang No. 1, respectively. These results show that the growth period of the Linlongxin series of strains that inherited the high-frequency clustered spikelet mutant sped1-D (L) gene is relatively short. When the shortest Linlongxin No. 1 is cultivated as the Guoqing rice stubble, its sowing start period is only 63 days (Table 3). The reason may be that SPED1 mutation leads to downregulation of the expression of GID1L2, a member of the gibberellin receptor family, resulting in the accumulation of endogenous gibberellin GA3 in cells (Jiang et al., 2014; Sui et al., 2012). Gibberellin is a highly effective and universal plant growth-promoting substance that can promote plant cell elongation, stem elongation, leaf expansion, accelerate growth and development, make crops mature earlier, and increase yield or improve quality (Li Baozhu et al., 2011; Drogoudi & Pantelidis, 2021).

[0109] The results of the agronomic trait investigation in this example show that the tillering rate, effective ear rate and ear formation rate of Linlongxin No. 1 and No. 2 cultivated as rice after wheat were higher than those of the control Yinxiang 38, while the tillering rate of Linlongxin No. 3 was lower than that of the control Yinxiang 38, but due to its high ear formation rate, the effective ears were still more than those of the control Yinxiang 38 (Table 4); when cultivated as National Day rice, these agronomic traits investigated after harvest were higher and lower between the Linlongxin series lines and the two controls (Table 4).

[0110] The results of this embodiment also show that, whether cultivated as wheat-rice stubble or National Day rice stubble, Linlongxin No. 1, No. 2 and No. 3 have significantly increased yields compared with the corresponding controls (Table 5). Compared with the National Day rice control, the increase in yield is 10-27% when cultivated as wheat-rice stubble and 24-65% when cultivated as National Day rice stubble; compared with the wheat-rice control, the increase in yield is 2-17% when cultivated as wheat-rice stubble and 10-46% when cultivated as National Day rice stubble (Table 5); corresponding to this, their spike length is longer, and the number of spikelets is not necessarily more than that of the wheat-rice control Yinxiang 38, which has a longer growth period of 9-23 days than them, but it is at least 20% more than the National Day rice control Songzaoxiang No. 1, which has a shorter growth period. These results show that the breeding of sped1-D (L) has a good yield-increasing effect while promoting crop maturity.

[0111] Thousand-grain weight is one of the important yield traits of rice. From the results in Table 4, it can be seen that the breeding of sped1-D(L) can obtain both the Linlongxin 1 and 2 varieties with small thousand-grain weight and the Linlongxin 3 variety with large thousand-grain weight. In addition, from the results in Table 4, it can be seen that the plant height of Linlongxin 1, 2 and 3 is between 85-96CM under the two crop rotation cultivation conditions, and the developed stems are thick and resistant to lodging.

[0112] Table 4 Agronomic performance of Linlongxin 1, 2 and 3 in two consecutive seasons of variety comparison trials (2023, Chongming, Shanghai)

[0113]

[0114]

[0115] Table 5 Yield performance of Linlongxin 1, 2 and 3 in two consecutive seasons of strain comparison trials (2023, Chongming, Shanghai)

[0116]

[0117] *: indicates that the yield increase reached a significant level; **: indicates that the yield increase reached an extremely significant level.

[0118] The results of the direct seeding yield comparison experiment of this embodiment are listed in Table 6. It can be seen from Table 6 that under the direct seeding conditions, the agronomic traits and yield performance of Linlongxin No. 1 are equally excellent. Although its growth period is the shortest, with a sowing period of only 63 days, the number of effective ears per unit area reached 209,600 ears, the fruit setting rate reached 86.61%, and the final yield was equivalent to 454.85 kg per mu, all of which were between the two controls, and at the same time had the characteristics of the longest ear length and the largest number of grains per ear. In addition, the thousand-grain weight of Linlongxin No. 1 was the same as that under artificial transplanting conditions, and the plant height increased by about 10 cm, but due to the thick development of the stems, its lodging resistance was not affected.

[0119] Example 6: Rice quality analysis and rice tasting identification of Linlongxin 1 derived from a high-frequency clustered spikelet mutant

[0120] In order to fully understand the impact of breeding high-frequency clustered spikelets sped1-D (L) on rice quality and rice texture and taste, the inventor sent samples of Linlongxin No. 1 to the Rice and Product Quality Supervision and Inspection Testing Center of the Ministry of Agriculture and Rural Affairs for measurement. The results obtained were: polished rice rate 82.6%, whole polished rice rate 60.3%, polished rice length 5.6mm, aspect ratio 2.3, chalky grain rate 17.0%, chalkiness 1.6%, transparency level 2, amylose content 15.0%, gelatin consistency 79mm, gelatinization temperature 5.0, protein content 8.2%, most of the rice quality indicators are at an excellent level, and no linkage traits with significant adverse effects on rice quality were found.

[0121] On this basis, the inventors used two soft rice varieties that are popular in the Shanghai market, which are also the control varieties used in the comparative test in Example 5, Songzaoxiang No. 1 and Yinxiang 38, as controls, and tasted and identified the Linlongxin No. 1 rice. The results are listed in Table 7.

[0122] As can be seen from Table 7, the comprehensive score of Linlongxin No. 1, including the scores of each sub-item such as smell, color, appearance, palatability and taste, is higher than Yinxiang 38, but lower than Songzaoxiang No. 1. The taste value measured by the HYM-1AC rice taste meter produced by Shanghai Huyueming Scientific Instrument Co., Ltd. is 80 points for Songzaoxiang No. 1 and 68 points for Linlongxin No. 1, which is 12 points lower, reflecting the actual situation that the amylose content of Linlongxin No. 1 is higher than that of Songzaoxiang No. 1 soft rice.

[0123] In addition, many users reported that the house was filled with fragrance when cooking rice with Linlongxin No. 1 rice, and they could taste the fragrance from the rice cooked with Linlongxin No. 1 rice.

[0124] Example 7 Identification of aroma components of Linlongxin 1 and 2 rice derived from high-frequency clustered spikelet mutants

[0125] In order to study the aroma source of the Linlongxin No. 1 rice of Example 6, the inventors prepared polished rice of Linlongxin No. 1 and Linlongxin No. 2, and sent the brown rice sample of Linlongxin No. 1 to Ingel Testing Technology Service (Shanghai) Co., Ltd. for gas chromatography (HS-SPME-GCMS) analysis of the flavor components contained therein. The results showed that the 2-acetyl-1-pyrroline aroma component contained in conventional fragrant rice (Thai fragrant rice and Wuchang fragrant rice, etc.) was not detected, but these aroma components were detected. The aroma components that are not commonly found in rice, such as Linlongxin No. 1 polished rice contains limonene, anethole, methyl myristate, galaxol, trimethyl borate, nonanal and benzaldehyde (Table 8); Linlongxin No. 2 polished rice contains limonene, anethole, methyl myristate, galaxol, trimethyl borate, nonanal, benzyl benzoate, mannitol and 2-(decanoyloxy)propane-1,3-diyl dioctanoate (Table 8), and its aroma is more fragrant than Linlongxin No. 1. The detection of these aroma components indicates that the aroma source may come from the sped1-D(L) mutant, because the male parent Lemont used to breed Linlongxin No. 1 and No. 2 does not carry the aroma gene.

[0126] Therefore, the inventors treated the grain powder samples of the sped1-D(L) mutant with 1.7% KOH and soaked them at room temperature for 10 minutes, and found that aroma was indeed produced. Then, we tasted the rice and proved that the sped1-D(L) mutant rice produced a strong aroma. After that, the inventors sent the rice samples of the sped1-D(L) mutant to Ingel Testing Technology Service (Shanghai) Co., Ltd. for gas chromatography (HS-SPME-GCMS) analysis of the flavor components contained in it. It was also found that it did not contain 2-acetyl-1-pyrroline, but contained limonene, anethole, methyl myristate, galaxol, trimethyl borate, nonanal and benzaldehyde, just like Linlongxin No. 1 and No. 2. These results indicate that the source of the aroma of the sped1-D(L) mutant and its derivatives Linlongxin No. 1 and No. 2 may be related to the mutation of SPED1 to sped1-D(L). Existing reports have shown that the expression of GID1L2, a member of the gibberellin receptor family, is downregulated in the sped1-D (L) allelic mutant sped1-D (Jiang et al., 2014). Other reports have shown that endogenous gibberellin GA3 accumulates in the GID1 mutant sdg (Sui et al., 2012). The accumulation of endogenous GA3 is likely to be similar to the effect of exogenous application of GA3, both of which can induce the synthesis of monoterpenes and sesquiterpenes in all tissues (Murcia et al., 2017). It is well known that most monoterpenes and sesquiterpenes are the main components of the lower boiling point part of volatile oils, and their oxygen-containing derivatives have higher boiling points, and most of them have strong aroma and physiological activity. Other induced synthesis include: sugars, aldehydes, sugar alcohols, organic acids and amino acids. It is precisely because of this that the sped1-D (L) mutant and its derivatives have the source of fragrance.

[0127]

[0128] Table 8 Results of flavor component detection of Linlongxin 1 and 2 rice derived from the high-frequency clustered spikelet mutant sped1-D(L)

[0129]

[0130]

[0131]

Claims

1. A mutant protein expressing high-frequency clustered spikelet traits sped1 -D(L) application, characterized in that The mutant protein sped1 -D(L) for use in regulating the growth period shortening trait of rice plants; The mutant protein sped1 The amino acid sequence of -D(L) is shown in SEQ ID NO:

1.

2. The mutant protein according to claim 1 sped1 -D(L) application, characterized in that The mutant protein sped1 -D(L), which exists in monomeric form.

3. An application of an isolated polynucleotide, characterized in that: The polynucleotide encodes the mutant protein according to any one of claims 1 to 2 sped1 -D(L), the mutant protein sped1 -D(L) is used for regulating the shortening of growth period of rice plants.

4. The use of the polynucleotide according to claim 3, characterized in that: The sequence of the polynucleotide is shown in SEQ ID NO:

2.

5. A method for cultivating clustered spikelet rice, characterized in that: The method comprises introducing the polynucleotide according to claim 4 into the target rice genome, and breeding heterozygous or homozygous genotypes expressing high-frequency clustered spikelet traits in the offspring, thereby obtaining clustered spikelet rice; The polynucleotide is introduced into the target rice genome through hybrid breeding, backcross breeding or gene editing technology, assisted by SNP markers at positions 226, 388 and 1341 in the sequence shown in SEQ ID NO: 2 or artificially developed CAPS marker selection; the CAPS marker includes using the SNP site at position 368 in the sequence shown in SEQ ID NO: 3 as the target site.

6. The method for cultivating clustered spikelet rice according to claim 5, characterized in that: The CAPS marker-assisted selection comprises the following steps: a) performing PCR amplification on a sample from a rice plant to be bred using the primers shown in SEQ ID NO: 4 and SEQ ID NO: 5; b1) performing restriction cleavage on the amplified product of step a) using a first restriction endonuclease, separating and detecting the length of the resulting cleavage product, wherein the first restriction endonuclease is selected from Asp26HI, Asp27HI, Asp35HI, Asp36HI, Asp40HI, Asp50HI, BsaMI, BscCI, BsmI, Uba1382I, Uba1415I, or Mva1269I; or b2) using a second restriction endonuclease to restrictively cut the amplified product of step a), separating and detecting the length of the resulting cut product, wherein the second restriction endonuclease is selected from BbsI, Bbv16II, BbvII, Bco102II, BsaVI, Bsc91I, BspBS31I, BspIS4I, BspVI, BstBS32I, BstTS5I, BsvTS514I, BpiI, or BpuAI; and c) breeding rice plants in which the SNP site is a C / A base.

7. A method for identifying rice with a high-frequency clustered spikelet trait, characterized in that: The method comprises detecting the polynucleotide according to claim 4 in a sample from the rice, wherein if the presence of a C / A base at the SNP site is detected, it indicates that the rice has a high-frequency clustered spikelet trait.

8. The mutant protein according to any one of claims 1 to 2 sped1 -D(L) or the polynucleotide of any one of claims 3-4 for use in rice breeding or germplasm resource improvement; expressing the mutant protein sped1 -D(L) or the rice of the polynucleotide is selected from the following traits: clustered spikelets with increasing frequency of occurrence; shortened reproductive period; and / or, The flavor profile does not contain 2-acetyl-1-pyrroline (2-AP).

9. The use according to claim 8, characterized in that The flavor profile includes limonene, anethole, methyl myristate, galaxol, trimethyl borate, nonanal, and benzaldehyde.

10. The use according to claim 8, characterized in that The flavor profile includes limonene, anethole, methyl myristate, galaxol, trimethyl borate, nonanal, benzyl benzoate, mannitol, and 2-(decanoyloxy)propane-1,3-diyl dioctanoate.

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