Allele of gene OsTZF5 for regulating and controlling thousand grain weight of rice and application of allele

Abstract

The invention relates to the field of crop genetic breeding, in particular to an allele of a gene OsTZF5 for regulating and controlling the thousand seed weight of rice and application of the allele. By regulating the allele (OsTZF5N) of the rice thousand seed weight gene OsTZF5 provided by the invention, the rice grain width and thousand seed weight can be enhanced. In the specific implementation of the invention, in order to explore the application value of the OsTZF5 gene in rice assisted breeding, the inventor analyzes genome data of 533 parts of rice varieties and finds that the OsTZF5 has two main haplotypes, namely Hap1 (OsTZF5N) and Hap2 (OsTZF59). Compared with Hap2, the expression level of Hap1 is lower. The invention further finds that the natural excellent allele OsTZF5N of the OsTZF5 gene can endow rice with larger grain width and thousand grain weight, and has important value for breeding high-yield rice varieties.

Description

Technical Field

[0001] The present invention relates to the field of crop genetic breeding, and particularly to alleles of the gene OsTZF5 that regulates the thousand-grain weight of rice and their applications. Background Art

[0002] Rice (Oryza sativa L.) is one of the important food crops globally, and its yield level is directly related to food security and the stable development of agricultural economy. Among the three elements of yield composition, the number of panicles per unit area, the number of grains per panicle, and the thousand-grain weight jointly determine the final yield of rice. Among them, the thousand-grain weight is an important indicator reflecting the grain size and the degree of filling, and has a significant impact on the rice yield. In recent years, as several genes related to yield, such as genes controlling grain size (GS3, SW5, and GW8), grain width and weight (GW2, GW5), genes for grain length and width (qGL1, qGW1, GS7, and qSS7), and the thousand-grain weight gene (TGW6), have been cloned successively, some genes related to yield have been widely used in the breeding of high-yield rice varieties.

[0003] As one of the three elements of rice yield, the thousand-grain weight has attracted much attention from breeders. During the conventional breeding process, the determination of the thousand-grain weight is generally carried out after the rice is mature. The whole process has a long cycle, a large sample size, and requires a lot of manpower. These factors increase the breeding cost and delay the breeding progress. Therefore, exploring the key genes that regulate the thousand-grain weight of rice and applying them to breeding practice have become one of the important strategies to improve rice yield. In recent years, scholars at home and abroad have carried out a large number of studies on the regulatory mechanism of rice thousand-grain weight, and initially constructed a theoretical framework for rice thousand-grain weight. Based on these theories, breeders have successfully cultivated a batch of rice new varieties with significantly increased thousand-grain weight, effectively improving the food yield. However, different alleles of the same gene may have significant differences in function. Mining natural alleles with a large thousand-grain weight phenotype can not only avoid the potential risks of transgenic technology, but also be directly applied to actual rice production. Existing studies have not specifically clarified the direct impact of the OsTZF5 gene on grain traits, such as grain size, thousand-grain weight, seed setting rate, or grain filling degree. In addition, there is currently no study providing excellent allelic variations of the OsTZF5 gene. Therefore, by mining excellent allelic variations of the OsTZF5 gene and other genes related to yield, it is an important way to further improve rice yield. Summary of the Invention

[0004] The object of the present invention is to provide alleles of the gene OsTZF5 that regulates the thousand-grain weight of rice and their applications to solve the problems existing in the above-mentioned prior art. The present invention provides excellent alleles of the rice OsTZF5 gene, which can be used to identify and cultivate rice varieties with a large thousand-grain weight, can promote the rice breeding process, and provide new gene resources for rice germplasm improvement.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides the application of the allele of the OsTZF5 gene in regulating rice yield traits, and the yield traits include grain width and / or 1000-grain weight; the nucleotide sequence of the allele is as shown in SEQ ID NO.1.

[0007] Preferably, the yield traits of rice containing the allele of the OsTZF5 gene are higher than those of rice without the allele of the OsTZF5 gene.

[0008] The present invention provides the application of the allele of the OsTZF5 gene in identifying rice yield traits, and the yield traits include grain width and / or 1000-grain weight; the nucleotide sequence of the allele is as shown in SEQ ID NO.1.

[0009] Preferably, the yield traits of rice containing the allele of the OsTZF5 gene are higher than those of rice without the allele of the OsTZF5 gene.

[0010] The present invention provides the application of the allele of the OsTZF5 gene in rice breeding, and the nucleotide sequence of the allele is as shown in SEQ ID NO.1.

[0011] The present invention provides the application of the allele of the OsTZF5 gene in cultivating high-yield rice, and the high-yield indicators include grain width and / or 1000-grain weight.

[0012] The present invention provides a method for increasing the grain width and / or 1000-grain weight of rice, including the step of crossing rice without the allele of the OsTZF5 gene with rice containing the allele of the OsTZF5 gene, so that the obtained hybrid rice contains the allele of the OsTZF5 gene; the nucleotide sequence of the allele is as shown in SEQ ID NO.1.

[0013] Preferably, the rice containing the allele of the OsTZF5 gene includes japonica rice.

[0014] Preferably, the japonica rice includes Nipponbare.

[0015] The present invention discloses the following technical effects:

[0016] The present invention provides a gene OsTZF5 related to rice grain width and 1000-grain weight 9 or its allele OsTZF5 N; combined with the phenotypic analysis of haplotypes and chromosome segment substitution lines, it was demonstrated that the OsTZF5 gene is related to grain width and 1000-grain weight. By regulating the alleles of the rice 1000-grain weight gene OsTZF5 provided by the present invention (OsTZF5 N is the natural excellent allele of OsTZF5), the grain width and 1000-grain weight of rice can be enhanced. In the specific implementation of the present invention, in order to explore the application value of the OsTZF5 gene in rice assisted breeding, the inventors analyzed the genomic data of 533 rice varieties and found that there are two main haplotypes of the OsTZF5 gene, Hap1 (OsTZF5 N ) and Hap2 (OsTZF5 9 ). The present invention found that the expression level of Hap1 is lower than that of Hap2. The present invention further found that the natural excellent allele OsTZF5 N of the OsTZF5 gene can endow rice with greater grain width and 1000-grain weight, which has important value for breeding high-yield rice varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0018] Figure 1 Characteristics and comparison of CCCH-type zinc finger proteins; among them, A shows the number of CCCH proteins and CCCH motifs in rice, barley, Medicago truncatula, maize, Arabidopsis thaliana, citrus, grape, switchgrass, wheat, soybean, rapeseed, turnip, tomato and poplar; B shows the proportion of CCCH proteins containing 1, 2, 3, 4, 5, 6, 7 CCCH motifs in rice, barley, Medicago truncatula, maize, Arabidopsis thaliana, citrus, grape, switchgrass, wheat, soybean, rapeseed, turnip, tomato and poplar; C shows the number of each CCCH motif in rice, barley, Medicago truncatula, maize, Arabidopsis thaliana, citrus, grape, switchgrass, wheat, soybean, rapeseed, turnip, tomato and poplar;

[0019] Figure 2 Phylogenetic relationship, motif composition and domain analysis of rice CCCH genes; among them, A shows the motifs predicted by MEME for OsC3H proteins, and different colors represent different motifs; B shows the domains of OsC3H proteins, and different colors are used to represent different domains;

[0020] Figure 3 Distribution and co-temporal relationship on rice chromosomes; among them, red genes represent segmentally duplicated genes, and blue boxes represent tandemly duplicated genes;

[0021] Figure 4 Phylogenetic relationship and synchronicity analysis of 14 plants; among them, A shows the evolutionary relationship of rice, barley, Medicago truncatula, maize, Arabidopsis thaliana, citrus, grape, switchgrass, wheat, soybean, rapeseed, turnip, tomato and poplar, and the number of homologous genes between the other 13 species and rice; B shows the synchronicity analysis between rice and Arabidopsis thaliana; C shows the possible synchronicity analysis between rice and maize;

[0022] Figure 5 The number of cis-regulatory elements in rice OsC3Hs; among them, different font colors represent different types of elements, and blue, green and red represent stress response, tissue-specific expression and plant growth regulators respectively;

[0023] Figure 6 The expression profile of OsC3Hs in different tissues;

[0024] Figure 7 The expression levels of the above-ground part and roots of rice after drought, MeJA and ABA treatments; among them, A shows the expression level in the stem; B shows the expression level in the root;

[0025] Figure 8 Identification of alleles of the rice OsTZF5 gene;

[0026] Figure 9 Phenotypic differences between two haplotypes of the rice OsTZF5 gene; among them, A shows the statistical chart of grain length of Hap1 and Hap2; B shows the statistical chart of grain width of Hap1 and Hap2; C shows the statistical chart of 1000-grain weight of Hap1 and Hap2;

[0027] Figure 10 The expression levels of two haplotypes of the rice OsTZF5 gene;

[0028] Figure 11 Phenotypic differences of substitution lines of the rice OsTZF5 gene; among them, A shows the phenotypic map of grain length of 9311 and CSSL1; B shows the phenotypic map of grain width of 9311 and CSSL1; C shows the statistical chart of grain length of 9311 and CSSL1; D shows the statistical chart of grain width of 9311 and CSSL1; E shows the statistical chart of 1000-grain weight of 9311 and CSSL1. Detailed implementation manners

[0029] The various exemplary implementation manners of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and implementation schemes of the present invention.

[0030] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0031] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0032] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the present invention specification are obvious to those skilled in the art. The present invention specification and examples are merely exemplary.

[0033] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0034] Experimental materials

[0035] Using 533 natural water varieties (http: / / ricevarmap.ncpgr.cn / ) and the indica rice 9311 (OsTZF5 9 ) as the recurrent parent (recipient parent), and the japonica rice NPB as the donor parent, CSSL1 (OsTZF5 N ) in the chromosome segment substitution lines was used as the experimental material. CSSL1 (OsTZF5 N ) was screened from the previously constructed CSSL population.

[0036] Example 1 Identification of different alleles of the OsTZF5 gene

[0037] 1. Identification of CCCH proteins in rice

[0038] The inventors retrieved the CCCH protein sequences in Arabidopsis thaliana from the Arabidopsis thaliana plant group database (https: / / phytozome-next.jgi.doe.gov / ), and identified the homologous CCCH proteins in rice (Oryza sativa) through a BLASTP search in the plant group, with an e-value cut-off of <1e -5 . To confirm the presence of the CCCH domain in the identified proteins, additional domain analyses were performed using multiple databases, including Pfam (PF 00642, http: / / pfam.xfam.org / ), SMART (SM 00356, http: / / smart.embl-heidelberg.de / ), InterPro (https: / / www.ebi.ac.uk / interpro / ), and the NCBI Conserved Domain Database (CDD, https: / / www.ncbi.nlm.nih.gov / cdd).

[0039] The rice CCCH proteins were subsequently renamed according to their chromosomal positions. The isoelectric point (pI), molecular weight (MW), and protein length of the OsC3H proteins were calculated using the ExPASy ProtParam tool. The subcellular localization of the OsC3H proteins was predicted using WoLF PSORT (https: / / wolfpsort.hgc.jp / ).

[0040] Through genome-wide identification and systematic analysis, the inventors identified 73 CCCH genes in the rice genome. These genes were systematically renamed from OsC3H1 to OsC3H73 in the order from chromosome 1 to 12 according to their chromosomal positions (Table 1). The bioinformatics characteristics encoding the OsC3H proteins showed considerable heterogeneity in physicochemical properties: the protein lengths ranged from 146 to 1890 amino acid residues (average = 535.9), the predicted isoelectric point (pI) was between 4.68 and 9.61, and the molecular weight showed a wide range from 15.8 kDa to 207.2 kDa. Subcellular localization prediction indicated that most CCCH proteins were enriched in the nucleus, with a few localized to other cellular compartments (Table 1). Subcellular localization prediction indicated that most OsC3H proteins were likely to be localized in the nucleus (Table 1).

[0041] Subsequently, the inventors further investigated the CCCH genes and motifs in 14 species, including rice, barley, Medicago truncatula, maize, Arabidopsis thaliana, citrus, grape, switchgrass, wheat, soybean, rapeseed, turnip, tomato, and poplar. Among them, the literature on switchgrass and wheat did not provide the CCCH motif types ( Figure 1)。 The number of CCCH genes in these species ranges from 34 to 155, and the corresponding motif counts range from 86 to 398. In rice, 160 CCCH motifs were identified in 73 genes, which is at an intermediate level among these 14 species.

[0042] The C3H gene family generally has 1 - 6 CCCH motifs. The number of CCCH motifs in each OsC3H protein was counted. Among them, 27 OsC3H proteins contain 1 CCCH motif, 24 proteins have 2 motifs, 13 have 3 motifs, 1 protein has 4 motifs, 6 have 5 motifs, and 2 have 6 motifs. This distribution is consistent with the trend observed in the other 13 species, where most CCCH proteins have 1 or 2 motifs, followed by 3 or 5 motifs. A total of 25 different types of CCCH motifs were identified in the 14 species. Among them, the two motifs - C - X8 - C - X5 - C - X3 - H and C - X7 - C - X5 - C - X3 - H are the most common and seem to be the ancestral forms of other CCCH motifs. In addition, rare motif types, such as C - X6 - C - X5 - C - X3 - H, were only found in specific species such as tomato and soybean. Notably, rice contains a unique motif, C - X8 - C - X5 - C - X4 - H, which was not found in any other species tested.

[0043] Table 1. Characteristics of the CCCH gene family in rice.

[0044]

[0045]

[0046] 2. Phylogenetic and protein structure analysis of OsC3H family members

[0047] Using the Pfam (PF00642), SMART (SM00356), InterPro, and NCBI CDD databases, the positions of the CCCH protein domain and other functional protein domains were determined. In addition, the MEME website was used to predict 10 conserved motifs of the OsC3H proteins. The MUSCLE method in the MEGA11 software was used to perform a multiple sequence alignment of the OsC3Hs proteins, and then a phylogenetic tree of OsC3Hs was constructed by the neighbor-joining method using the protein sequences, with the bootstrap value set to 1000. The same method was used to construct an evolutionary tree of the CCCH proteins in rice and Arabidopsis to evaluate the reliability of the branches. The phylogenetic tree of the CCCH proteins in rice (Oryza sativa) and Arabidopsis was constructed using the same method. The presence and positions of the CCCH domain and other functional protein domains were verified through multiple databases, including the Pfam (PF00642), SMART (SM00356), InterPro, and the NCBI Conserved Domain Database (CDD). To identify the conserved motifs in the OsC3H proteins, the inventors used the MEME suite, setting the number of motifs to 10.

[0048] To investigate the evolutionary relationships and structural features of the CCCH proteins, the inventors conducted a comprehensive phylogenetic and structural analysis using the protein sequences of Arabidopsis and rice. The inventors performed a multiple sequence alignment and constructed a phylogenetic tree. According to the established classification criteria for Arabidopsis CCCH proteins, the rice CCCH proteins can be divided into 11 different subgroups (designated as I-XI). There were significant differences in the number of genes within each subgroup. To further investigate the evolutionary relationships within the rice CCCH gene family, the inventors constructed a phylogenetic tree based on 73 OsC3H protein sequences using MEGA software. In addition, motif and domain analyses were performed using MEME and multiple functional annotation databases. The results showed that members within the same subgroup exhibited highly conserved motif compositions and domain distributions ( Figure 2 ). Notably, in addition to the typical CCCH domain, we also found multiple functional domains in these proteins. For example, the subgroup VIII members OsC3H16, OsC3H41, and OsC3H66 were found to contain the RNA recognition motif (RRM) domain. In addition, other subgroups contained proteins with additional functional domains, including the WD40, ANK, and KH domains. These findings suggest that the CCCH domain may act in concert with other domains, potentially expanding the functional diversity and regulatory complexity of CCCH proteins in various biological processes.

[0049] 3. Genome localization, gene duplication, and synteny analysis of the OsC3H family

[0050] Visualize the OsC3H family genes using TBtools. Analyze the synteny relationships between the rice genome and rice and other plant species using the MCScanX plugin in TBtools.

[0051] To estimate the evolutionary rates of the identified genes, calculate the ratio of non-synonymous substitutions (Ka) to synonymous substitutions (Ks) using KaKs_Calculator 3.0. The Ka / Ks ratio is an indicator of the selection pressure acting on a gene.

[0052] Chromosomal localization analysis of OsC3H genes shows that their distribution in the rice genome is significantly uneven. The most OsC3H genes are in chromosome 1 (12 genes), followed by 10 genes in chromosome 6. In contrast, chromosomes 10 and 11 each contain only one OsC3H gene, with the lowest distribution density ( Figure 3 ). Gene duplication is the main driving force for the expansion of plant gene families and plays a key role in functional innovation and adaptive evolution. In the examples, the inventors identified 8 pairs of duplicated gene pairs in the rice CCCH gene family, including 1 pair of tandem duplication pairs (OsC3H23 gene / OsC3H24 gene) and 7 pairs of segmental duplication pairs. Phylogenetic analysis shows that these duplicated gene pairs cluster within the same subfamily or clade. For example, the OsC3H2 gene / OsC3H38 gene is classified into subfamily XI. To evaluate the selection pressure acting on these duplicated OsC3H gene pairs, the inventors calculated their Ka / Ks ratios. The Ka / Ks values of all 8 pairs of duplicated gene pairs are less than 1, indicating the action of purifying selection and suggesting limited functional divergence after the duplication event. In addition, the inventors also studied the synteny relationships between rice and 13 other species, including 9 monocots and 4 dicots ( Figure 4 ). The analysis shows that the number of homologous gene pairs between rice and the nine dicot species ranges from 2 to 19 (specifically: 7, 12, 15, 12, 19, 4, 2, 2, and 2), while the number of homologous gene pairs between rice and the four monocot species ranges from 58 to 167 (specifically: 58, 167, 81, and 107) ( Figure 4 ). The number of collinear gene pairs between rice and dicots is 7, 12, 15, 12, 19, 4, 2, 2, and 2 respectively. In contrast, the number of collinear gene pairs between rice and monocots is significantly higher, being 58, 167, 81, and 107 pairs respectively ( Figure 4 ). This difference indicates that the level of synteny between rice and dicots is lower compared to rice and monocots, a finding consistent with the phylogenetic relationships between these species.

[0053] 4. Promoter analysis of CCCH genes in rice

[0054] Extract the 2-kb upstream sequence of the OsC3H gene from the rice (Oryza sativa) genome using TBtools. The extracted sequence was analyzed for cis-regulatory elements using the PlantCARE database.

[0055] The inventors analyzed the cis-regulatory elements in the promoter region (2000 bp upstream of the start codon) of the OsC3H gene and identified a total of 22 functional cis-regulatory elements ( Figure 5 ). Among them, light-responsive elements were the most abundant, including the majority of the predicted elements ( Figure 5 ). In addition, the inventors also identified 11 hormone-responsive elements, including auxin-responsive elements (TGA element, AuxRR-core, AuxRE, and TGA-box), gibberellin-responsive elements (TATC-box, GARE motif, and P-box), salicylic acid-responsive element (tca element), and MeJA-responsive elements (CGTCA-motif and TGACG-motif). Notably, abscisic acid (ABA) and methyl jasmonate (MeJA) responsive elements were the most prominent, occurring 432 and 644 times, respectively. Various regulatory elements related to biotic and abiotic stresses were also observed in the promoter region of the OsC3H gene. A total of 339 anaerobic induction elements, including 205 AREs and 134 gc-motifs, were detected in 66 OsC3H genes. 85 low-temperature response elements (LTRs) were identified in 47 genes, and 128 drought-responsive elements were identified in 58 genes. The inventors also identified six cis-regulatory elements related to plant organogenesis. These genes included the meristem expression-related CATbox (53 genes), the zein metabolism regulatory o2 site (53 genes), and the endosperm expression-related GCN4 motif (10 genes). These results indicate that OsC3Hs play important roles in regulating rice growth and development, mediating hormone signal transduction, and responding to various biotic and abiotic stresses.

[0056] 5. Expression analysis of OsC3H genes

[0057] Download the expression levels of OsC3H genes under different tissues, MeJA treatment, JA treatment, and drought treatment from the PPRD database (http: / / ipf.sustc.edu.cn / pub / plantrna / ). Using the data of hormone treatment and drought treatment as materials of 14-day germinated seedlings, extract RNA after 3 h, 6 h, 12 h, and 24 h, respectively. Normalize the gene expression levels using log10(PFKM + 1) and create a heatmap of the gene expression levels.

[0058] Analysis of tissue-specific expression profiles provides important insights into the potential functions of genes in plants. Based on the expression data of six rice tissues, we observed different expression patterns of OsC3H genes ( Figure 6 ). Genes in Group I showed high expression levels in all six tissues, while genes in Group II showed moderate expression levels. In contrast, most genes in Group III were low or not expressed in these tissues, although some showed significant tissue-specific expression. For example, the OsC3H10 gene was mainly expressed in seeds, while the OsC3H37 gene was specifically expressed in flowers. These findings suggest that different OsC3H genes may be involved in the development and functions of specific tissues in rice. In addition to tissue-specific expression, we also analyzed the responses of OsC3H genes to drought stress and hormonal treatments with abscisic acid (ABA) and methyl jasmonate (MeJA). Expression levels in stem and root tissues were measured at six time points (0 h, 1 h, 3 h, 6 h, 12 h, and 24 h) respectively ( Figure 7 ). Under drought conditions, after 24 h, the expression levels of the OsC3H5, OsC3H38, and OsC3H40 genes in the stem increased by 10.71-fold, 9.05-fold, and 3.47-fold, respectively. In the root, the OsC3H10 gene showed a significant increase, by 28.52-fold, although it was hardly detectable in the stem. Similarly, during MeJA treatment, after 24 h, the expression of the OsC3H40 gene also showed a significant increase, by 22.12-fold and 21.96-fold in the stem and root, respectively. In addition, the expression level of the OsC3H32 gene in the root increased by 45.98-fold. After 24 h of ABA treatment, the expression of the OsC3H40 gene increased by 3-fold in the stem and 16-fold in the root. These results indicate that certain OsC3H genes are highly sensitive to drought, ABA, and MeJA stresses, but their expression patterns vary among different tissues. This highlights their potential role in mediating stress responses and promoting tissue-specific adaptation mechanisms in rice.

[0059] 6. Haplotype analysis of OsC3Hs genes

[0060] Based on the high expression levels of the OsC3H36 gene in multiple tissues, the inventors selected it for haplotype analysis. Currently, there are relevant literatures naming the OsC3H36 gene as the OsTZF5 gene. Therefore, in the following, the OsC3H36 gene will also be referred to as the OsTZF5 gene in this invention. The specific steps of the haplotype analysis are as follows: The inventors downloaded the SNP genotyping data of 533 different rice varieties from the database (http: / / ricevarmap.ncpgr.cn / ) and performed haplotype analysis on the SNPs of the OsTZF5 gene. SNPs in the 2 kb upstream region, the OsTZF5 gene itself, and the 1 kb downstream region of the OsTZF5 gene were selected for haplotype analysis, and two major haplotypes were screened out for further analysis. The results are as Figure 1 shown. The results showed that the OsTZF5 gene was mainly divided into two haplotypes, Hap1 (OsTZF5 N ) and Hap2 (OsTZF5 9 ), with a total of 29 SNP differences. Among them, 20 SNPs were located upstream of the gene, 1 SNP was located in the 5' UTR region, 5 SNPs were located in the CDS region, and 3 SNPs were located downstream of the gene ( Figure 8 ). It is worth noting that the Hap1 haplotype mainly exists in japonica rice varieties, while the Hap2 haplotype is mainly associated with indica rice varieties. Currently, there are no reports on different alleles of the OsTZF5 gene. Therefore, this invention reports different alleles of the OsTZF5 gene for the first time.

[0061] Among them, OsTZF5 N

[0062] OsTZF5 9

[0063] Example 2 Analysis of Phenotypes and Expression Levels of Different Haplotypes

[0064] The inventors downloaded data on grain length, grain width, and 1000-grain weight of 533 rice samples from the database (https: / / ricevarmap.ncpgr.cn / ), and used the t-test to determine whether there were significant differences in expression levels and phenotypes among different haplotypes (P < 0.05). The results are as Figure 9 and Figure 10 shown. The analysis found that there was no significant difference in grain length between Hap1 (SEQ ID NO.1) and Hap2 (SEQ ID NO.2), but compared with Hap2, the grain width and 1000-grain weight of Hap1 were significantly increased ( Figure 9 ); in addition, the expression level of Hap1 was significantly higher than that of Hap2 ( Figure 10 ).

[0065] The above examples show that the OsTZF5 gene plays a functional role in regulating these agronomic traits. Specifically, phenotypes such as grain width and 1000-grain weight can be changed by appropriately regulating the expression of alleles of the OsTZF5 gene, thereby affecting rice yield ( Figure 9 ).

[0066] Example 3 Phenotype Analysis of Chromosome Segment Substitution Lines

[0067] The inventors screened chromosome segment substitution lines with 9311 as the recipient parent (recurrent) and NPB as the donor parent from the CSSL population established in the early stage of the laboratory. That is, 9311 was used as the recipient parent as the recurrent parent for 5 generations of hybridization to obtain chromosome segment substitution lines. After that, materials matching the haplotype of the OsTZF5 gene (named CSSL1 (OsTZF5 N )) or CSSL1 were screened. After harvesting, the grain length and width of 20 seeds were measured, and the 1000-grain weight was measured 5 times, and then the t-test (P < 0.05) was performed. The specific statistical method: sowing and harvesting were carried out according to the conventional steps. After the rice was completely mature, the seeds were dried in an incubator at 37°C, and measured with a vernier caliper and a ten-thousandth balance. The results are as Figure 11 shown. The results showed that there was no significant difference in grain length between CSSL1 (OsTZF5 N genotype) and 9311 (OsTZF5 9 genotype), while the grain width and 1000-grain weight of CSSL1 were significantly higher than those of 9311, further confirming that OsTZF5 9 and its allele OsTZF5 N have a new function of regulating rice grain width and 1000-grain weight, and the excellent allelic variation OsTZF5 NIt can increase the grain width and 1000-grain weight of rice.

[0068] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention should fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of the allele of OsTZF5 gene in regulating rice yield traits, characterized in that: The yield trait includes grain width and / or thousand-grain weight; the nucleotide sequence of the allele is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: The yield trait of rice containing the allele of the OsTZF5 gene is higher than that of rice not containing the allele of the OsTZF5 gene.

3. Application of the allele of OsTZF5 gene in identifying rice yield traits, characterized in that: The yield trait includes grain width and / or thousand-grain weight; the nucleotide sequence of the allele is shown in SEQ ID NO.

1.

4. The use according to claim 3, characterized in that: The yield trait of rice containing the allele of the OsTZF5 gene is higher than that of rice not containing the allele of the OsTZF5 gene.

5. Application of the allele of OsTZF5 gene in rice breeding, characterized in that: The nucleotide sequence of the allele is shown in SEQ ID NO.

1.

6. The use of the allele of OsTZF5 gene in cultivating transgenic rice, characterized in that: The nucleotide sequence of the allele is shown in SEQ ID NO.

1.

7. A method for increasing rice grain width and / or thousand-grain weight, characterized in that: The method comprises the steps of hybridizing rice without the allele of the OsTZF5 gene and rice containing the allele of the OsTZF5 gene, so that the hybridized rice contains the allele of the OsTZF5 gene; the nucleotide sequence of the allele is shown in SEQ ID NO.

1.

8. The method according to claim 7, characterized in that The rice containing the allele of the OsTZF5 gene includes japonica rice.

9. The method according to claim 8, characterized in that The japonica rice includes Nipponbare.

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