Polymorphic site and application thereof in regulation and control of dip angle of rice leaf

Abstract

The invention provides a polymorphic site and application of the polymorphic site in regulation and control of a dip angle of a rice leaf. The invention discloses a single nucleotide polymorphism (SNP) for identifying leaf dip angle phenotypes or an sf0623501417 site of rice, the sf0623501417 site is located in an IAA23 gene, and a corresponding basic group is T or G. The sf0623501417 site can be applied to prediction or analysis of plant type compactness, close planting property and / or lodging resistance and photosynthesis efficiency of rice (population); the method can be applied to rice germplasm resource identification, directional breeding and breeding of rice and the like. The invention provides a new direction for optimized breeding of rice.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant breeding; more specifically, the present invention relates to a polymorphic site and its application in regulating the inclination angle of rice leaves. Background Art

[0002] Rice plant traits (such as leaf inclination, plant height, number of tillers, etc.) have an important impact on photosynthesis, population density, lodging resistance, etc. during rice growth. In particular, leaf inclination, as a key plant trait, directly affects the efficiency of rice light energy utilization and the maximization of photosynthesis.

[0003] The inclination angle of rice leaves refers to the angle between the rice leaves and the vertical direction, which is used to describe the degree of inclination of the leaves of rice plants. Under normal circumstances, rice leaves should grow vertically, but in actual growth, affected by factors such as light, wind, and nutrition, the rice leaves may have a certain degree of inclination. The leaf inclination angle can be detected using some common measuring tools, such as angle rulers, laser instruments, etc. Common methods include: (a) Angle ruler method: Use an angle ruler to measure the angle between the rice leaves and the vertical direction. (b) Laser instrument method: Use a laser instrument to emit a laser beam, measure the angle between the laser beam and the rice leaves, and then calculate the size of the leaf inclination angle.

[0004] A moderate leaf inclination angle can enable rice to absorb more sunlight, increase the efficiency of photosynthesis, and increase yield; while a leaf inclination angle that is too large or too small will lead to insufficient light energy capture or uneven light energy distribution, affecting photosynthesis and reducing yield. Therefore, reasonable control of rice leaf inclination angle is one of the keys to optimizing rice cultivation management. The formation of rice leaf inclination angle is related to many factors, mainly including the following aspects: (a) Light conditions: Adequate light can promote the normal growth of rice leaves and reduce the occurrence of leaf inclination angle. (b) Wind factors: Strong winds can cause rice plants to tilt, resulting in an increase in leaf inclination angle. (c) Nutrition supply: Adequate nitrogen fertilizer supply can promote the increase in rice plant height and reduce the occurrence of leaf inclination angle. (d) High temperature stress: Under high temperature environment, rice leaves are prone to baking, resulting in an increase in leaf inclination angle.

[0005] Rice leaf inclination is an important indicator of the growth and development of rice plants and has a significant impact on rice yield. Through reasonable regulation methods and technologies, the size of rice leaf inclination can be effectively controlled to ensure the normal growth and development of rice plants. Although traditional breeding methods such as hybrid breeding and mutation breeding have made some progress, these methods have problems such as long cycle, low efficiency, and narrow genetic basis.

[0006] In summary, the leaf inclination angle of rice is regulated by multiple genes. For the research on the leaf inclination angle of rice, the field currently lacks a technology that can accurately regulate the leaf inclination angle at the single base level, and further in-depth research and exploration is needed. Summary of the invention

[0007] The purpose of the present invention is to provide a polymorphic site and its application in regulating the inclination angle of rice leaves.

[0008] In a first aspect of the present invention, a method for identifying (including predicting) a rice leaf inclination phenotype is provided, comprising targeting a single nucleotide polymorphism (SNP) or haplotype at the sf0623501417 site of rice (corresponding to position 238 in the sequence shown in SEQ ID NO: 1); if the site is base T, the rice has a small leaf inclination (relatively small) and a compact plant type (relatively compact); if the site is base G, the rice has a large leaf inclination (relatively large).

[0009] In one or more embodiments, if the leaf inclination angle of the rice is small, it indicates that the plant type is compact (relatively compact), and the rice is a densely planted and / or lodging-resistant type.

[0010] In one or more embodiments, if the leaf inclination angle of the rice plant is large, it indicates that the plant type is expanded (relatively expanded).

[0011] In one or more embodiments, the analysis of single nucleotide polymorphism (SNP) or haplotype adopts a method selected from the following group: sequencing method, PCR amplification method, probe method, hybridization method, chip method, restriction enzyme analysis method, allele polymorphism analysis method, and electrophoresis method.

[0012] In one or more embodiments, the reagents or devices for specifically detecting single nucleotide polymorphisms or haplotypes may include: chips, probe sets, primer sets, primer probe sets, restriction enzymes, electrophoresis reagents, and gene sequencing instruments.

[0013] In another aspect of the present invention, the use of the rice sf0623501417 site or the IAA23 gene containing the site is provided, for: as a target for identifying (including predicting) the rice leaf tilt phenotype; or, for preparing a reagent, device or kit for identifying (including predicting) the rice leaf tilt phenotype; wherein, during the identification, the single nucleotide polymorphism or haplotype of the rice sf0623501417 site is analyzed; or, when preparing the reagent, the reagent is targeted at the single nucleotide polymorphism or haplotype of the sf0623501417 site.

[0014] In another aspect of the present invention, a reagent for specifically analyzing the rice sf0623501417 site or the IAA23 gene containing the site is provided for identifying the rice leaf inclination phenotype; wherein the reagent is directed to the single nucleotide polymorphism or haplotype of the sf0623501417 site.

[0015] In one or more embodiments, the identification is carried out by analyzing the single nucleotide polymorphism or haplotype of the sf0623501417 site; if the site is base T, the rice has a small leaf inclination angle and a compact plant shape (relatively small); if the site is base G, the rice has a large leaf inclination angle (relatively large).

[0016] In one or more embodiments, the rice sf0623501417 locus is used as a target for (but not limited to) predicting or analyzing the compactness, density and / or lodging resistance, and photosynthesis efficiency of rice (population) by analyzing the rice leaf inclination phenotype; identifying rice germplasm resources; or conducting directed breeding and selection of rice.

[0017] In one or more embodiments, the reagents or devices include (but are not limited to): primers (sets) for detecting the region containing the rice sf0623501417 site, probes (sets), primer probe sets, chips, restriction enzymes for distinguishing the base types of the sf0623501417 site, electrophoresis reagents, and gene sequencing instruments.

[0018] In one or more embodiments, the nucleotide sequence of the IAA23 gene is as shown in SEQ ID NO: 1, wherein corresponding to the sf0623501417 site (position 238), the base type includes base T or base G.

[0019] In one or more embodiments, the amino acid sequence of the protein encoded by the IAA23 gene is as shown in SEQ ID NO: 1, wherein the amino acid residue corresponding to the sf0623501417 position (position 80) includes S or A.

[0020] In another aspect of the present invention, a method for optimizing rice breeding is provided, comprising: a rice variety with a small leaf inclination angle (such as ZH11 or NIP varieties) is known, or the leaf inclination angle of rice is analyzed and identified by any of the methods described above, and when the sf0623501417 site is base T, the rice variety is identified as having a small leaf inclination angle; for the rice variety with a small leaf inclination angle, the T at the sf0623501417 site is mutated to base G, thereby regulating the increase of its leaf inclination angle (thereby promoting the expansion of its plant type and regulating its photosynthesis efficiency).

[0021] In another aspect of the present invention, a method for optimizing rice breeding is provided, comprising: identifying a rice variety with a large leaf inclination angle (such as the DHY rice variety), or analyzing and identifying the leaf inclination angle of rice by any of the methods described above, and identifying the rice variety with a large leaf inclination angle when the sf0623501417 site is a base G; for the rice variety with a large leaf inclination angle, mutating the G at the sf0623501417 site to a base T, thereby regulating the leaf inclination angle to decrease (thereby promoting a compact plant type, promoting close planting and / or lodging resistance, and regulating its photosynthesis efficiency).

[0022] In one or more embodiments, the method for modification includes (but is not limited to) the following techniques: site-directed mutagenesis, gene editing (such as CRISPR / Cas technology) or homologous recombination; or regulating the expression of the rice IAA23 gene in rice, wherein the sf0623501417 site of the rice IAA23 gene is selected from base T or base G.

[0023] Other aspects of the present invention will be apparent to those skilled in the art in view of the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The leaf inclination angle of the gene-edited material of IAA23 with ZH11 as the background (iaa23-cr / ZH11) was reduced, while the leaf inclination angle of its overexpression material (IAA23-OE / ZH11) was significantly increased.

[0025] Figure 2 There are 4 SNPs in the nucleotide sequence of IAA23 that are highly correlated with leaf inclination angle.

[0026] Figure 3 There are 4 SNPs in the nucleotide sequence of IAA23 that are highly correlated with leaf inclination angle.

[0027] Figure 4 , SNPs are strongly associated with the distribution in the maximum and minimum leaf inclination populations.

[0028] Figure 5 , leaf inclination phenotype analysis of SNP endogenous replacement materials. DETAILED DESCRIPTION

[0029] After extensive research, the inventors have revealed for the first time a single nucleotide polymorphism (SNP) or haplotype target for identifying leaf inclination phenotypes. The SNP is located at the sf0623501417 site of rice, which belongs to the site in the IAA23 gene, and the corresponding base is T or G. The sf0623501417 site can be used to predict or analyze the compactness, dense planting and / or lodging resistance, and photosynthesis efficiency of rice (population); it can be used to identify rice germplasm resources, carry out directional breeding and selection of rice, etc. The present invention provides a new direction for optimized breeding of rice.

[0030] In the prior art of this field, technicians have not had a clear understanding of which gene or genes control the rice leaf tilt phenotype, and the prior art lacks a gene target for identifying the rice leaf tilt phenotype. Based on the inventor's new discovery, the present invention provides a single nucleotide polymorphism or haplotype target suitable for identifying the rice leaf tilt phenotype, namely, the sf0623501417 locus of rice. The present invention also provides a target gene suitable for identifying the rice leaf tilt phenotype, namely, IAA23.

[0031] In the present invention, unless otherwise specified, the IAA23 may be a gene having a nucleotide sequence as shown in SEQ ID NO: 1 (from rice), wherein the SNP is present at a specific position. The present invention also includes sequence variants having the same function as IAA23 (but the SNP sites focused on by the present invention are conserved).

[0032] According to the new discovery of the present invention, a method for identifying the leaf inclination phenotype of rice is provided, comprising using the single nucleotide polymorphism (SNP) or haplotype of the rice at the sf0623501417 site as a target; if the site is the base T, the leaf inclination of the rice is small and the plant type is compact (relatively small); if the site is the base G, the leaf inclination of the rice is large (relatively large).

[0033] According to the new discovery of the present invention, those skilled in the art can use any of the various techniques known in the art or being developed to analyze nucleic acid sequences, and these techniques can all be included in the present invention. The methods described include, but are not limited to: sequencing, PCR amplification, probe method, hybridization, restriction enzyme analysis, allele polymorphism analysis (such as melting curve method) to identify nucleic acid sequences, etc.

[0034] Genome-wide association studies (GWAS) can also be applied to analyze the SNP sites. Genome-wide association studies are a technique for studying the association between genotype and phenotype by detecting genetic markers, such as SNPs, in multiple individuals across the genome, thereby identifying genetic variants associated with target traits.

[0035] The method for obtaining DNA from the sample to be tested is a technique well known to those skilled in the art, for example, the traditional phenol / chloroform / isoamyl alcohol method can be adopted, or some commercially available DNA extraction kits can be used, such kits are well known to those skilled in the art.

[0036] The polymerase chain reaction (PCR) technique is well known to those skilled in the art, and its basic principle is a method for in vitro enzymatic synthesis of specific DNA fragments. The method of the present invention can be carried out using conventional PCR technology.

[0037] According to the above, the present invention also relates to a kit for identifying the leaf inclination phenotype of rice, wherein the kit contains a reagent for specifically detecting the SNP of interest or a specific gene containing the SNP site of the present invention. In some optional ways, the kit may also include: a restriction endonuclease that can recognize the changed position of the SNP site. It should be understood that according to the content disclosed by the present invention, the reagents that can be used for identification are not limited thereto.

[0038] In addition, the kit may also contain instructions for use and / or standard operating procedures for identification. The kit can achieve the purpose of rapid detection and batch detection of rice leaf inclination phenotypes.

[0039] The present invention overcomes the current situation of lack of tools for identifying rice leaf inclination phenotype. Based on the target provided by the present invention and combined with the existing knowledge in the field, a simple and effective identification method and its preferred primers can be designed, thereby providing a feasible method for early identification or rapid identification of rice leaf inclination phenotype and providing a powerful tool for plant breeding screening.

[0040] Being able to identify the rice leaf inclination phenotype in the early stages of planting or before planting can greatly facilitate plant breeding.

[0041] After knowing the role of the sf0623501417 site or IAA23 containing the key site, it can be used as a molecular marker to carry out targeted screening of plants. It is also possible to screen for substances or potential substances that can target and regulate the rice leaf tilt phenotype by changing / regulating the protein at the key site in IAA23 based on this new discovery.

[0042] Methods for screening substances acting on a gene or protein or a specific region thereof as a target are well known to those skilled in the art, and these methods can be used in the present invention. The candidate substances can be selected from: peptides, polymeric peptides, peptidomimetics, non-peptide compounds, carbohydrates, lipids, antibodies or antibody fragments, ligands, organic small molecules, inorganic small molecules and nucleic acid sequences, etc. Depending on the type of substance to be screened, those skilled in the art will know how to select an appropriate screening method.

[0043] With respect to the SNP or specific gene of interest disclosed in the present invention, through large-scale screening, a class of potential substances that specifically act on the IAA23 gene or protein and have a regulatory effect on the rice leaf inclination phenotype can be obtained.

[0044] The present invention also provides a method for optimizing rice breeding. In a preferred embodiment, for a rice with a relatively small leaf inclination angle (such as NIP variety), the T at the sf0623501417 site is mutated to base G, thereby regulating its leaf inclination angle to increase, which is beneficial to promoting its plant type expansion, regulating its photosynthesis efficiency, and achieving more efficient production.

[0045] As another preferred embodiment of the present invention, any of the above-mentioned methods is used to analyze and identify the inclination angle of rice leaves. When the sf0623501417 site is base T, it is identified as a small leaf inclination angle. The T at the sf0623501417 site is mutated into base G, thereby regulating the increase of the leaf inclination angle, which is beneficial to promote the expansion of the plant type, regulate its photosynthesis efficiency, and achieve more efficient production.

[0046] As another preferred embodiment of the present invention, for a rice with a relatively large leaf inclination angle (such as the Rice Flower Fragrance Yangzhou (DHY) variety), the G at the sf0623501417 site is mutated into a base T, thereby regulating its leaf inclination angle to decrease, thereby promoting its compact plant type, promoting close planting and / or lodging resistance, regulating its photosynthesis efficiency, and achieving more efficient production.

[0047] As another preferred embodiment of the present invention, any of the methods described above is used to analyze and identify the inclination angle of rice leaves. When the sf0623501417 site is the base G, it is identified as a large leaf inclination angle. The G at the sf0623501417 site is mutated into the base T, thereby regulating the leaf inclination angle to decrease, thereby promoting a compact plant type, promoting close planting and / or lodging resistance, regulating the photosynthesis efficiency, and achieving more efficient production.

[0048] It is understood that any method applicable to targeted base / amino acid variation can be applied to the present invention. For example, the following techniques can be used, including but not limited to: site-directed mutagenesis, gene editing (such as CRISPR / Cas technology) or homologous recombination. As a preferred method, after clarifying the key regulatory genes and key sites of rice plant type traits such as leaf inclination angle, it is preferred to use gene editing technology to accurately modify specific genes or sites, so as to improve rice plant type traits such as leaf inclination angle more efficiently and accurately.

[0049] In the present invention, the full-length or functional fragments of the IAA23 gene can also be used to implement the transformation, including regulating the expression of the rice IAA23 gene in rice.

[0050] As a preferred embodiment of the present invention, when the sf0623501417 site of the rice IAA23 gene is base T (the amino acid residue at the corresponding site of the encoded amino acid is S) and the leaf inclination angle of the rice needs to be increased, the expression of the IAA23 gene whose sf0623501417 site is base G (the amino acid residue at the corresponding site of the encoded amino acid is A) can be regulated in the rice (such as by gene editing and replacement).

[0051] As another preferred embodiment of the present invention, when the sf0623501417 site of the rice IAA23 gene is base G (the amino acid residue at the corresponding site of the encoded amino acid is A) and the leaf inclination angle of the rice needs to be reduced, the expression of the IAA23 gene whose sf0623501417 site is base T (the amino acid residue at the corresponding site of the encoded amino acid is S) can be regulated in the rice (such as by gene editing and replacement).

[0052] In summary, the SNP markers related to rice leaf inclination and other plant traits of the present invention can promote the molecular marker-assisted selection of rice leaf inclination, effectively accelerate the rice breeding process, improve breeding efficiency, and have important significance for rice breeding. The present invention has application prospects in the molecular design breeding of rice leaf inclination phenotypes and the use of genetic engineering technology to improve crop varieties.

[0053] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually carried out according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning Experiment Guide, Science Press, or according to the conditions recommended by the manufacturer.

[0054] Example 1: Target gene screening

[0055] After extensive screening, genomic analysis and experimental analysis, the inventors screened out a candidate gene IAA23 related to rice leaf inclination. The gene sequence and amino acid sequence are as follows:

[0056] Gene sequence of IAA23 (position 238 is T) (SEQ ID NO: 1):

[0057] ATGTCGACGAGCTCCGGCGCCGACTCGTCCCCGCCCGTCTCGGGCCTCGACTACGACGACCGCCCTCACCCTCGCCCTCCCCGGCTCCTCCTCCTCCTCCTCCTCCACCGCCGACCCCGAACGCAAGCGCCGCCCACGCCGACCACGCCGACGCCAAGCCGCCTTCCCCAAAGGCGCGGGCGGTGGGGTGGCCGCCGGTGAGGGCGTACAGGCGGAACGCCTGCGGGAGGAC T CCGCGAGGGCGAAGCTGGTCAAGGTGGCCGTGGACGGCGCGCCGTACCTGCGCAAGGTGGACCTCGCGGCGCACGCCGGCTACGCGCCCCTCCTCCGCGCTCCACGCATGTTCGCCTCCTGCCTCGCCGTCCGCGGCGGCGGCGGCGGCGACGGCGAGGGTACAAAGCT CGTCGACTTGGTCACCGGCGCCGAGTACGTGCCCACCTACGAGGACAAGGACGGCGACTGGATGCTCGTCGGCGACGTCCCCTGGAAGATGTTCGTCGAGTCCTGCAAGCGGATCCGTCTCATGAAGAGCTCCGAGGCCGTCAACTTATCGCCAAGACGATCATCCAGATAA

[0058] The amino acid sequence of IAA23 (position 80 is S) (SEQ ID NO: 2):

[0059] MSTSSGADSSPPVSGLDYDDTALTLALPGSSSSSSSTADPERKRAAHADHADAKPPSPKARAVGWPPVRAYRRNALRED S ARAKLVKVAVDGAPYLRKVDLAAHAGYAPLLRALHGMFASCLAVRGGGGGDGEGTKLVDLVTGAEYVPTYEDKDGDWMLVGDVPWKMFVESCKRIRLMKSSEAVNLSPRRSSR*

[0060] IAA23 gene editing vector: Using CRISPR / Cas9 gene editing technology, gRNA is introduced into the CRISPR / Cas9 vector to obtain a recombinant vector for gene editing.

[0061] gRNA sequence: CCCCGGCTCCTCCTCCTCCTCCT (SEQ ID NO: 3)

[0062] IAA23 gene overexpression vector: The gene sequence of IAA23 was introduced into the pCAmbia 1300 vector to obtain a recombinant vector for overexpression.

[0063] Using rice ZH11 as the background, the above expression vector was used to construct transgenic materials, and IAA23 knockout gene editing materials (iaa23-cr / ZH11) and IAA23 overexpression materials (IAA23-OE / ZH11) were obtained. Rice was planted and genetic phenotypes were observed and analyzed.

[0064] The results showed that the leaf inclination angle of the IAA23 gene-edited material (iaa23-cr / ZH11) with ZH11 as the background was significantly reduced. In contrast, the leaf inclination angle of the IAA23 overexpression material (IAA23-OE / ZH11) was significantly increased ( Figure 1 ).

[0065] Therefore, it is clear that the IAA23 gene is involved in the regulation of rice leaf inclination.

[0066] Example 2: Analysis of Single Nucleotide Polymorphism (SNP) Related to IAA23 Rice Leaf Inclination

[0067] Considering the possible role of IAA23 in rice plant type domestication and differentiation between indica and japonica rice, single nucleotide polymorphisms (SNPs) associated with rice leaf inclination of IAA23 were analyzed.

[0068] The results of bioinformatics analysis showed that four SNPs may be highly correlated with leaf inclination, namely: sf0623501049, sf0623501417, sf0623501964 and sf0623502256. Figure 2 shown.

[0069] Further analysis revealed that the four SNPs were significantly differentiated between indica and japonica rice. Figure 2 shown.

[0070] Among them, two SNPs are in the exon region of the gene, involving changes in amino acids ( Figure 3 ), respectively as follows:

[0071] sf0623501417S80->A;

[0072] sf0623501964I->R。

[0073] IAA23 M The gene sequence of IAA23 (the 238th position is G) (SEQ ID NO:4):

[0074] ATGTCGACGAGCTCCGGCGCCGACTCGTCCCCGCCCGTCTCGGGCCTCGACTACGACGACACCGCCCTCACCCTCGCCCTCCCCGGCTCCTCCTCCTCCTCCTCCTCCACCGCCGACCCCGAACGCAAGCGCGCCGCCCACGCCGACCACGCCGACGCCAAGCCGCCTTCCCCAAAGGCGCGGGCGGTGGGGTGGCCGCCGGTGAGGGCGTACAGGCGGAACGCGCTGCGGGAGGAC G CCGCGAGGGCGAAGCTGGTCAAGGTGGCCGTGGACGGCGCGCCGTACCTGCGCAAGGTGGACCTCGCGGCGCACGCCGGCTACGCGCCCCTCCTCCGCGCGCTCCACGGCATGTTCGCCTCCTGCCTCGCCGTCCGCGGCGGCGGCGGCGGCGACGGCGAGGGTACAAAGCTCGTCGACTTGGTCACCGGCGCCGAGTACGTGCCCACCTACGAGGACAAGGACGGCGACTGGATGCTCGTCGGCGACGTCCCCTGGAAGATGTTCGTCGAGTCCTGCAAGCGGATCCGTCTCATGAAGAGCTCCGAGGCCGTCAACTTATCGCCAAGACGATCATCCAGATAA

[0075] IAA23 M The amino acid sequence of IAA23 (the 80th position is A) (SEQ ID NO:5):

[0076] MSTSSGADSSPPVSGLDYDDTALTLALPGSSSSSSSTADPERKRAAHADHADAKPPSPKARAVGWPPVRAYRRNALRED AARAKLVKVAVDGAPYLRKVDLAAHAGYAPLLRALHGMFASCLAVRGGGGGDGEGTKLVDLVTGAEYVPTYEDKDGDWMLVGDVPWKMFVESCKRIRLMKSSEAVNLSPRRSSR*

[0077] Example 3: Relationship between SNP or haplotype of IAA23 and rice leaf inclination

[0078] The inventors further compared the IAA23 SNP with the leaf inclination angle and sequence of 378 japonica rice samples and found that its haplotype distribution in the maximum and minimum leaf inclination angle populations had a strong correlation ( Figure 4 ).

[0079] Through the sequence determination and analysis of some varieties, it was found that the distribution pattern of the second SNP (sf0623501417S80->A) was significant, specifically, the second SNP in the population with the largest leaf inclination was G (about 100%), and the second SNP in the population with the smallest leaf inclination was T (about 100%), such as Figure 4 shown.

[0080] Example 4: Functional verification of SNP of IAA23

[0081] In order to further verify the function of the second SNP, the inventors selected small-leaf inclination materials (Nipponbare, NIP) and large-leaf inclination materials (Daohuaxiang Yangzhou, DHY) as the transformation background, and replaced the second SNP in the inner circle, specifically, in NIP, T was replaced by G; and in DHY, G was replaced by T. According to the target gene and the SNP site to be replaced, the gRNA sequence related to the endogenous replacement was designed, and it was ensured to have high specificity to avoid off-target effects. The designed gRNA sequence was obtained by gene synthesis technology and cloned into a specific CRISPR vector.

[0082] gRNA sequence: CCCCGGCTCCTCCTCCTCCTCCT (SEQ ID NO: 3)

[0083] After the vector is constructed, the CRISPR system and gRNA vector are transformed into rice recipient cells together using the Agrobacterium-mediated method to modify the target gene. After transformation, the gene editing effect is verified by PCR, sequencing and other methods to ensure the mutation or knockout of the target gene. By culturing and screening rice seedlings, the successfully edited plants are selected for further sequence analysis to obtain the gene editing material with the correct SNP replacement.

[0084] The successfully edited rice was planted in the field, and plant traits such as leaf inclination were measured and analyzed. The results showed that in the NIP with a small inclination angle, the leaf inclination angle increased after replacing T with G.

[0085] In the DHY with large inclination angle, after G is replaced by T, the blade inclination angle also tends to decrease significantly, such as Figure 5 shown.

[0086] Therefore, by comparing the phenotypic changes in leaf inclination before and after editing, it can be clearly seen that the SNP of IAA23 has a regulatory effect on rice plant type traits.

[0087] Summarize:

[0088] The present invention successfully improves the plant type traits of rice by precisely regulating key genes related to the leaf inclination of rice. By regulating the leaf inclination, rice can better utilize light energy, improve photosynthesis efficiency, and thus increase yield. In addition, by improving the plant type, the lodging resistance of rice is also enhanced, and the adaptability is stronger. The method of the present invention has high efficiency, accuracy and operability, and has broad application prospects.

[0089] The present invention is applicable to rice breeding and improvement, and has important application value in increasing rice yield, optimizing rice cultivation mode, and improving rice lodging resistance, etc. The technology can be promoted and applied in rice breeding enterprises, agricultural research institutions, and agricultural production.

[0090] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims. At the same time, all the documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference separately.

Claims

1. A method for identifying the leaf inclination phenotype of rice, comprising using the single nucleotide polymorphism or haplotype of the sf0623501417 site of rice as a target; if the site is base T, the leaf inclination of the rice is small and the plant type is compact; if the site is base G, the leaf inclination of the rice is large.

2. The method according to claim 1, characterized in that If the leaf inclination angle of rice is small, it indicates that the plant is compact and is a densely planted and / or lodging-resistant type of rice; If the leaf inclination angle of rice is large, it indicates that the plant shape is expanding.

3. The method according to claim 1, characterized in that The analysis of single nucleotide polymorphism or haplotype adopts a method selected from the group consisting of sequencing, PCR amplification, probe method, hybridization method, chip method, restriction enzyme analysis, allele polymorphism analysis, and electrophoresis; or Reagents or devices for specifically detecting single nucleotide polymorphisms or haplotypes may include: chips, probe sets, primer sets, primer probe sets, restriction enzymes, electrophoresis reagents, and gene sequencing instruments.

4. Use of the rice sf0623501417 locus or the IAA23 gene containing the locus as a target for identifying the rice leaf tilt phenotype; or, for preparing a reagent, device or kit for identifying the rice leaf tilt phenotype; in, During identification, the single nucleotide polymorphism or haplotype of the rice sf0623501417 site is analyzed; or, during preparation of the reagent, the reagent is targeted at the single nucleotide polymorphism or haplotype of the sf0623501417 site.

5. Use of a reagent for specifically analyzing the rice sf0623501417 locus or the IAA23 gene containing the locus for identifying the rice leaf inclination phenotype; wherein, The reagent targets the single nucleotide polymorphism or haplotype of the sf0623501417 site.

6. The use according to any one of claims 4 or 5, characterized in that The identification is carried out by analyzing the single nucleotide polymorphism or haplotype of the sf0623501417 site; if the site is base T, the rice has a small leaf inclination angle and a compact plant type; if the site is base G, the rice has a large leaf inclination angle.

7. The use according to any one of claims 4 or 5, characterized in that The rice sf0623501417 locus is used as a target to predict or analyze the compactness, dense planting and / or lodging resistance, and photosynthesis efficiency of rice by analyzing the rice leaf inclination phenotype; Conduct rice germplasm resource identification; or conduct directional breeding and selection of rice; Preferably, the reagents or devices include: primers for detecting the region containing the rice sf0623501417 site, probes, primer-probe sets, chips, restriction enzymes for distinguishing the base types of the sf0623501417 site, electrophoresis reagents, and gene sequencing instruments.

8. The use according to any one of claims 4 or 5, characterized in that The nucleotide sequence of the IAA23 gene is shown in SEQ ID NO: 1, wherein corresponding to the sf0623501417 site, the base type includes base T or base G; The amino acid sequence of the protein encoded by the IAA23 gene is shown in SEQ ID NO: 1, wherein the amino acid residue corresponding to the sf0623501417 position includes S or A.

9. A method for optimizing rice breeding, comprising: (a) A rice plant having a small leaf inclination angle is known, or the leaf inclination angle of the rice plant is analyzed and identified by the method described in any one of claims 1 to 3, and when the sf0623501417 site is a base T, the rice plant having a small leaf inclination angle is identified; for the rice plant having a small leaf inclination angle, the T at the sf0623501417 site is mutated to a base G, thereby regulating the leaf inclination angle to increase; or (b) A rice variety with a large leaf inclination angle is known, or the leaf inclination angle of the rice variety is analyzed and identified by the method described in any one of claims 1 to 3, and when the sf0623501417 site is the base G, the rice variety is identified as having a large leaf inclination angle; for the rice variety with a large leaf inclination angle, the G at the sf0623501417 site is mutated to the base T, thereby regulating the leaf inclination angle to decrease.

10. The method according to claim 9, characterized in that The transformation method includes the following technologies: site-directed mutagenesis, gene editing or homologous recombination; or regulating the expression of rice IAA23 gene in rice, wherein the sf0623501417 site of the rice IAA23 gene is selected from base T or base G.

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