Tartary buckwheat semi-dwarf high-yield gene sdw1 and SNP molecular marker and application thereof

Abstract

This invention discloses a gene for semi-dwarf buckwheat with high fertility. sdw1 This invention relates to the field of biotechnology, specifically to the SNP molecular markers and applications of buckwheat, and utilizes EMS to induce mutagenesis in the tartary buckwheat variety "Guiku No. 1". A semi-dwarf, high-fruiting mutant was discovered in the mutagenized offspring. Further research revealed that this semi-dwarf, high-fruiting trait is controlled by a single recessive nuclear gene. Through gene localization, mutation site genotyping, determination of endogenous gibberellin content, and exogenous gibberellin application, it was determined that this gene encodes the first key rate-limiting enzyme in the gibberellin biosynthesis pathway, endoroot-Cuba pyrophosphate synthase (CPS), which was named... sdw1 This invention discovers sdw1 The gene is an SNP mutant gene. This mutation can significantly increase the seed setting rate while reducing the height of tartary buckwheat plants, and it has no effect on other agronomic traits. It is genetically stable under different genetic backgrounds and has great utilization value in the breeding of high-yield and lodging-resistant tartary buckwheat.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a gene for semi-dwarf, high-fruiting buckwheat. sdw1 Its SNP molecular markers and applications. Background Technology

[0002] Tartary buckwheat is an important specialty grain in my country. Its seeds are rich in bioactive flavonoids, which have extremely high health benefits and play an important role in my country's health industry. However, tartary buckwheat not only has a low seed setting rate and low yield (the national average yield is 80 kg per mu), but also has a relatively tall plant height (1.2 m-1.5 m). It is extremely susceptible to large-scale lodging during continuous rainy weather, causing severe yield reduction or even crop failure. Furthermore, increasing planting density cannot increase yield, seriously affecting the healthy development of the tartary buckwheat industry. Therefore, strengthening the breeding and promotion of tartary buckwheat varieties that combine semi-dwarf stalks, lodging resistance, and high seed setting and yield is the most effective measure to solve the problem of low tartary buckwheat yield.

[0003] Plant height is a complex regulatory process regulated by numerous genes. Currently, over a hundred plant height-related genes have been cloned in plants and crops (Mohan et al., 2021; Cheng et al., 2022). However, apart from a few superior mutant genes used in the "Green Revolution," most mutations in plant height-related genes result in a significant decrease in yield when plant height is reduced. Therefore, creating and discovering new, superior dwarf genes that do not affect or even increase fruit setting are of great importance for breeding high-yielding, lodging-resistant crops.

[0004] Dwarf mutants are ideal materials for important genetic and gene biological function studies. Currently, some dwarf mutants in tartary buckwheat have been reported (Ma et al., 2019; Sun et al., 2019; Sun et al., 2021; Peng et al., 2022; Fang et al., 2023), but these dwarf mutants all cause a significant decrease in seed setting rate and yield while dwarfing the plant. No mutants or genes have been reported that can both reduce plant height and increase seed setting rate and yield. The current lack of gene resources that can reduce plant height without affecting seed setting, which can serve as a target for improvement, not only makes it difficult to meet the needs of tartary buckwheat molecular breeding but also limits the research progress on the synergistic regulation mechanism of tartary buckwheat plant height and yield. Therefore, discovering semi-dwarf, high-seed-setting tartary buckwheat mutants and cloning their regulatory genes is of great significance for revealing the molecular mechanism of tartary buckwheat plant height and for the breeding practice of high-yield and lodging-resistant tartary buckwheat. Summary of the Invention

[0005] The purpose of this invention is to provide a semi-dwarf, high-fruiting gene for tartary buckwheat. sdw1This invention, along with its SNP molecular markers and applications, aims to address the problems existing in the aforementioned prior art. For the first time, this invention identifies a gene in buckwheat that regulates plant height and seed setting rate. sdw1 The study discovered that a single-base SNP mutation in the exon leads to the substitution of the encoded amino acid, resulting in a semi-dwarf, high-fruiting phenotype. A molecular marker based on this SNP was developed, which can accurately identify the plant height and fruit setting rate of tartary buckwheat, and is of great significance for molecular breeding of high-yield and lodging-resistant tartary buckwheat.

[0006] To achieve the above objectives, the present invention provides the following solution: The present invention provides an sdw1 protein, the amino acid sequence of which is shown in SEQ ID NO.4 or SEQ ID NO.10.

[0007] This invention provides a semi-dwarf, high-fruiting gene in tartary buckwheat that encodes the aforementioned sdw1 protein. sdw1 The nucleotide sequence of the buckwheat semi-dwarf high-fruiting gene sdw1 is shown in SEQ ID NO.3 or SEQ ID NO.9.

[0008] This invention provides the above-mentioned sdw1 protein or the above-mentioned tartary buckwheat semi-dwarf high-fruiting gene. sdw1 Application in the cultivation of semi-dwarf, high-fruiting tartary buckwheat.

[0009] This invention provides a molecular marker related to the height and seed setting rate of buckwheat plants. The nucleotide sequence of the molecular marker is shown in SEQ ID NO.8, and a G / A mutation exists at the 61st base of the sequence.

[0010] The present invention provides primer pairs for amplifying the above-mentioned molecular markers, the primer pairs comprising the nucleotide sequences SNaPshot-sdw1 F as shown in SEQ ID NO.5, SNaPshot-sdw1R1 as shown in SEQ ID NO.6, and SNaPshot-sdw1 R2 as shown in SEQ ID NO.7.

[0011] This invention provides the above-mentioned molecular markers or primer pairs for the preparation of products used to identify the height and seed setting rate of tartary buckwheat plants.

[0012] This invention provides a product for identifying the height and seed setting rate of buckwheat plants, the product comprising the primer pair described above.

[0013] This invention provides the use of the above-described molecular markers, primer pairs, or products in any of the following: (1) Identify the plant height and seed setting rate of buckwheat; (2) Screening for semi-dwarf, tall-fruiting bitter buckwheat; (3) Molecular breeding of buckwheat; (4) Improvement of buckwheat varieties.

[0014] This invention provides a method for identifying buckwheat plant height and seed setting rate, comprising the following steps: Using the genomic DNA of the buckwheat to be tested as a template, PCR amplification was performed on the template using the primer pairs mentioned above. The plant height and seed setting rate of the buckwheat to be tested were determined by the genotype of the PCR amplification results. If the genotype of the buckwheat to be tested is GG or GA, then the buckwheat to be tested is a tall-stalked buckwheat with a low seed setting rate; if the genotype of the buckwheat to be tested is AA, then the buckwheat to be tested is a semi-dwarf buckwheat with a high seed setting rate.

[0015] This invention provides the application of reagents for editing the SDW1 gene in the cultivation of semi-dwarf, high-fruiting buckwheat, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0016] The present invention discloses the following technical effects: This invention utilizes EMS to mutate the buckwheat variety "Guiku No. 1". A semi-dwarf, high-fruiting mutant was discovered in the mutagenesis progeny. This mutant exhibits semi-dwarf stalks, thick stems, dark green leaves, and high fruit production. Further research revealed that this semi-dwarf, high-fruiting trait is controlled by a single recessive nuclear gene. Through gene localization, mutation site genotyping, determination of endogenous gibberellin content, and exogenous gibberellin application, it was determined that this gene encodes the first key rate-limiting enzyme in the gibberellin biosynthesis pathway, endoroot-cube pyrophosphate synthase (CPS), and is named... sdw1 This invention discovered sdw1 The gene is an SNP mutant gene. This mutation significantly increases the seed setting rate while reducing the plant height of tartary buckwheat, without affecting other agronomic traits. It is genetically stable under different genetic backgrounds and has great application value in breeding high-yield and lodging-resistant tartary buckwheat. A pair of SNaPshot primers were designed based on the SNP mutation site of this gene. These primers can be used to quickly identify or screen whether tartary buckwheat plants contain the gene. sdw1 Gene testing is reliable and simple, and its application as a genetic marker in molecular breeding for high-yield and lodging-resistant buckwheat has high application value. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 For wild parent Guiku No. 1 (WT) and sdw1 Mutant phenotype; where A represents WT and sdw1 Mutant phenotype; B represents WT and sdw1 Mutant intersegmental phenotype; C represents WT and sdw1 Field lodging resistance of mutants; Figure 2 For wild parent Guiku No. 1 (WT) and sdw1 Intersegmental longitudinal sections (A and B) and transverse sections (C and D) of the mutant and their cell length / width statistics; Figure 3 It is a semi-dwarf, high-fruiting mutant of buckwheat. sdw1 Gene localization; Figure 4 for sdw1 Genotyping based on gene variant sites; Figures 5-7 For wild parent Guiku No. 1 (WT) and sdw1 Analysis of gibberellin content in mutant strains; Figure 8 Spraying exogenous gibberellins on Guiku No. 1 (WT) and sdw1 The effect of mutant plant height; Figure 9 for sdw1 Allelic mutants sdw1-2 Phenotype (A) and content of three active gibberellins in stem (B); Figure 10 For overexpression sdw1 Genes and their wild-type genes SDW1 The effect of (non-mutated) on Arabidopsis plant height; where A is the phenotype and B is the statistical bar chart. Detailed Implementation

[0019] Various exemplary embodiments 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 rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0021] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0022] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0023] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0024] The gene SDW1, which regulates the plant height and seed setting of tartary buckwheat, Ft07.g34998 The gene, whose nucleotide sequence is shown in SEQ ID NO.1, is as follows:

[0025] The amino acid sequence of the SDW1 protein encoded by the gene SDW1 is shown in SEQ ID NO.2, as follows: .

[0026] Buckwheat semi-dwarf high-fruiting gene sdw1 Its nucleotide sequence is shown in SEQ ID NO.3, as follows: sdw1 The wild-type parental base at 2320bp was separated from the wild-type parental base, which changed the G base to the A base, causing the C base (lowercase part) at 2320bp of the coding sequence to change to the T base.

[0027] Buckwheat semi-dwarf high-fruiting gene sdw1 The amino acid sequence of the encoded sdw1 protein is shown in SEQ ID NO.4, as follows: MCTQSSSITLSLSFNTSSLPPSPSVLSIVAREKQFGRGLRRLRCNATPNPDNPSIQEIKKEAFGVLQSDRIPLVKWPNHDHNKDNLQIPTHNLKGNVLDKNNERVDEIRSILRSMNDGEISVSPYDTAWVALVKYINGKPQFSTSLEWIAKNQLEDGSWGDKDMFEAHDRIMNTLACVIALRTWNMCLNQCNKGMEFIRENLSKLEEEKDEHMPIGFEVVFPSLIEIARDLGIEFLNSGFLQHIHAQRNIKLQRIPKEILHNIPTSLLHSLEGMAGLEWEKLLKLQNKDGSFLFSPASTAFALMQTRDLNCLRYLENVVSKFNGGVPNVYPVDLFEHIWAVDRLQRLGISRFFKDEINECVNYVHRYWTENGICWARNSDVQDIDDTAMGFRLLRLHGYKVSPGVFKNFKKNGEFFCFAGQSTQAVTGMYNLLRASQLQYPGEEILEEARNFSSKYLIEKQACNELFDKWIIMKDLPGEVGYALDLPWYASLPRIESRLYVEQYGGDDDVWIGKTLYRMPYVNNNLYLDLAKLDFNACQDVHVLEWEGVRRWYEDYNLGEYGLSQDKLLRLYFVAAASIFEPERSTERLAWAKAAALVETIASHFSKQKDQEAFAHEFLNSCKSLDSSNNERKGILSTLLSTLNQLSMDVMVAHGVDIRDNLHQVWKKWMVSYSATGDRYTGEAELLTQTIQLANPNFGGLTSIGNDYQHLVDMTNKICHQLGHFQTTKHNTNQDMEVVVPNEVNEDMQRLAEMVFSSNDMDTNTKIGFWEVAwSYYYNTYCSATTIDAHIAKVMFERVVASS, at the 774th amino acid residue W (lowercase part) of this sequence, it is mutated from the amino acid residue R at the corresponding position in the wild-type SDW1 protein.

[0028] This invention involves ethyl methanesulfonate (EMS) mutagenesis of the high-yielding and widely adaptable buckwheat variety "Guiku No. 1". In the mutagenesis progeny, a genetically stable semi-dwarf mutant was discovered. This mutant exhibits characteristics such as plant height approximately 70% of the wild parent, thicker stems, darker green leaves, and a significantly increased seed setting rate. Further research revealed that the phenotype of this mutant is controlled by a single recessive nuclear gene, which was named semidwarf 1 (sdw1). This invention is based on this unexpected discovery.

[0029] Example 1 1. Tartary buckwheat semi-dwarf tall-fruiting mutant sdw1 Phenotypic analysis In this embodiment, EMS was used to induce mutagenesis in the buckwheat variety "Guiku No. 1" to obtain a semi-dwarf, high-fruiting mutant. sdw1 (Semi-dwarf, tall, sturdy mutant) sdw1 Published in the document "The complete reference genome ofTartary buckwheat and its mutation library provide important resources for genetic studies and breeding" (Hongyou Li, Qiuyu Lv, Taoxiong Shi, YalingJian, Bin Ran, Yuanzhi Cheng, Lei Wang, Jing Zhang, Juan Huang, Jiao Deng, Liwei Zhu, Qijiao Chen, Fang Cai, Ruiyuan Li, Qi Wu, Yizhong Zhang, YuliangZhang, Zhang Zhang, FengYu, Qingfu Chen. The complete reference genome ofTartary buckwheat and its mutation library provide important resources for genetic studies and breeding. Cell Reports, 2025, 44: 115621), provided by Guizhou Normal University).

[0030] For planting in multiple locations in Guizhou, Hainan and Sichuan sdw1Mutant identification showed that the mutant exhibited stable phenotypic inheritance, with a semi-dwarf stalk and high seed setting rate. Its plant height was approximately 70% of the wild parent "Guiku No. 1," with no change in the number of main stem nodes, shortened internodes (an average shortening of 28.37% compared to the wild type), significantly increased stem diameter (an average increase of 48.63% compared to the wild type), and significantly increased seed setting rate (an average increase of 58.44% compared to the wild type). It also possessed extremely strong lodging resistance. Figure 1 ).right sdw1 Paraffin sections were taken from the third internode of the mutant. The results showed that the longitudinal cell length of sdw1 was significantly shorter and the transverse cell length was significantly wider, consistent with its phenotype of shortened internodes and significantly increased stem diameter. Figure 2 ).

[0031] 2. Genetic analysis of the sdw1 semi-dwarf, high-fruiting mutant of tartary buckwheat. Using the wild parent "Guiku No. 1" as the male parent, sdw1 The mutant was used as the maternal parent for hybridization, resulting in four F1 hybrid plants. All F1 plants exhibited tall stature and normal fruit setting, suggesting... sdw1 The semi-dwarf, tall-fruiting phenotype of the mutant is likely controlled by a recessive gene. Further analysis of the F2 segregating population revealed 293 segregating plants, of which 210 exhibited the tall phenotype and 83 exhibited the dwarf phenotype, with a segregation ratio of approximately 2.53:1 (χ²). 2 =1.72<3.84), sdw1 The semi-dwarf tall fruit-bearing trait is controlled by a pair of recessive nuclear genes (Table 1).

[0032] Table 1 sdw1 Genetic characteristics of mutants 3. Gene of semi-dwarf, high-fruiting mutant buckwheat sdw1 Positioning Using the MutMap strategy, the wild parent 'Guiku No. 1' and sdw1 BSA sequencing was performed on the mutant and two extreme pools of the F2 generation. Using a ∆SNP index ≥ 0.6 as a threshold, the control... sdw1 The gene for the semi-dwarf, high-fruiting trait in the mutant is located in a 658.36 kb region on chromosome 7 (14560087-15218452). Figure 3 Analysis of this interval revealed that it contains a total of 5 SNP sites, all of which originated from previous studies on the M4 generation. sdw1 EMS-induced SNPs were identified through resequencing. Further annotation of these five SNP sites revealed that only one SNP (14,981,429) occurred in an exon of a gene (Table 2). At the genomic DNA level, this SNP is located in the gene... Ft07.g34998 On the 14th exon (located on the negative strand), in sdw1 In the mutant, the SNP changes from a G base to an A base in the wild-type parent, resulting in a C base to a T base at 2320 bp of the coding sequence, and consequently, a change from arginine (R) to tryptophan (W) at amino acid position 774. Ft07.g34998 Functional annotation of the gene revealed that it is a key enzyme gene for the first step of gibberellin synthesis in Arabidopsis thaliana—gibberellin synthesis endogenous-Cuba pyrophosphate synthase (GBS). CPS It is a homologous gene of ), therefore it is determined that this gene is sdw1 Candidate mutant genes.

[0033] Table 2 sdw1 Candidate SNPs for mutant genetic mapping and annotation 4. Tartary buckwheat semi-dwarf high-fruiting gene sdw1 Molecular markers and primer design against Ft07.g34998 To investigate the difference in the genomic sequence of this SNP between tall, normally fruiting and semi-dwarf, tall-fruiting plants, a pair of SNaPshot primers, SNaPshot-sdw1, was designed. The primer sequences are as follows: SNaPshot-sdw1 F: 5′-CTTTGGCAATGTGAGCATCG-3′ (SEQ ID NO.5); SNaPshot-sdw1 R1: 5'- GAAGGTGACCAAGTTCATGCT GTTTTGGGAGGTGGCGc-3′ (SEQ IDNO.6); SNaPshot-sdw1 R2: 5'- GAAGGTCGGAGTCAACGGATTG GTTTTGGGAGGTGGCGt-3′ (SEQ ID NO. 7).

[0034] In the primer sequences above, SNaPshot-sdw1 F is the universal upstream primer, SNaPshot-sdw1 R1 and SNaPshot-sdw1 R2 are the downstream primers, and the underlined part is the universal fluorescent adapter sequence; the 5' end of SNaPshot-sdw1 R1 is connected to the FAM fluorescent group, and the 5' end of SNaPshot-sdw1 R2 is connected to the HEX fluorescent group; the lowercase part at the 3' end is the genotyping site sequence.

[0035] The target fragment to be amplified is: CTTGGCAATGTGAGCATCGATGGTGGTAGCAGAACAATATGTATTGTAGTAATAACTCCRCGCCACCTCCCAAAAC (SEQ ID NO.8), where "R" indicates that the base at this site is G or A.

[0036] 5. Tartary buckwheat semi-dwarf high-fruiting gene sdw1 Verification of the effectiveness of molecular markers and primers Using SNaPshot primer SNaPshot-sdw1 ( sdw1 All 293 individual plants from the mutant × “Guiku No. 1” F2 population were subjected to quantitative real-time PCR amplification. The amplification system for quantitative real-time PCR was as follows: 5 μL of 2×PARMS master mix, 0.4 μL of upstream universal primer F (10 μM), 0.15 μL of downstream primer R1 (10 μM), 0.15 μL of downstream primer R2 (10 μM), 100 ng of DNA template, and ddH2O to a final volume of 10 μL. The DNA template was extracted from the leaves of individual buckwheat plants using the CTAB method. The PCR amplification program was as follows: 94℃ for 20 min; 94℃ for 20 s, 65-57℃ (-0.8℃ / cycle) for 1 min, cycle 10; 94℃ for 20 s, 57℃ for 1 min, cycle 32.

[0037] Genotyping was performed based on the collected signals. Blue fluorescent signals near the X-axis represented the GG genotype, green fluorescent signals near the Y-axis represented the AA genotype, and green fluorescent signals in the middle represented the GA genotype. The results are as follows: Figure 4 As shown. Genotyping results showed that among the 210 plants with the phenotype of tall stem and low fruit set (average stem height 115.4 cm, average fruit set rate 35.33%), there were two genotypes: GG and GA, with 72 plants of the GG genotype and 138 plants of the GA genotype; while all 83 plants with the semi-dwarf, high fruit set phenotype (average stem height 76.2 cm, average fruit set rate 55.98%) were of the AA genotype (…). Figure 4 There were 83 plants. Genotype GG:GA:AA = 1:1.92:1.15 ≈ 1:2:1 (χ²) 2 =1.70<χ 2 0.05 =5.99), indicating that the mutation site co-segregates with the phenotype, and genetic evidence shows that this mutation site caused... sdw1 The mutant exhibits a semi-dwarf, tall, and robust phenotype.

[0038] 6. Tartary buckwheat high-fruiting, semi-dwarf gene sdw1 Functional verification (1) sdw1Analysis of endogenous gibberellin content in mutants Further analysis Ft07.g34998 Does gene mutation cause this by affecting endogenous gibberellin levels? sdw1 The mutant exhibits a semi-dwarf, tall, and fruitful phenotype. This invention utilizes chromatography-mass spectrometry to determine the relationship between the wild parent, Guiku No. 1, and... sdw1 The content of 18 gibberellins in the 3rd-4th stem nodes of the mutant at the four-true-leaf stage was analyzed, and 11 gibberellins were detected. Figures 5-7 Compared to Guiku No. 1, sdw1 The mutant showed significantly reduced levels of biologically active GA1, GA4, and key GA synthesis precursors GA19, GA20, and GA44, indicating that... Ft07.g34998 Gene mutations lead to the inhibition of gibberellin synthesis, which in turn causes a lack of active gibberellin, inhibits stem cell elongation, and ultimately results in the dwarf and high-fruiting phenotype of sdw1 mutant plants.

[0039] (2) Exogenous application of gibberellin to... sdw1 Analysis of the effect of mutant plant height To further analyze whether exogenous gibberellin application can restore [the condition]... sdw1 The plant height of the mutants, based on the Guiku No. 1 at the one-true-leaf stage and sdw1 Using mutant seedlings as material, exogenous spraying with 75 mg / L GA3 was applied, with the spraying amount determined by dripping from the plant leaves. A control group treated with distilled water was also included. After 15 days of treatment, in the control group treated with distilled water... sdw1 The mutant plants were significantly shorter than those of Guiku No. 1; while in the experimental group sprayed with gibberellin... sdw1 The mutant's plant height recovered to the level of Guiku No. 1. Figure 8 This indicates that exogenous GA3 can restore... sdw1 The plant height of the mutant sdw1 The mutant is a gibberellin-sensitive mutant.

[0040] (3) sdw1 Verification of gene allelic mutations Through analysis of resequencing data from the previously constructed mutant library, this embodiment found that... Ft07.g34998 A novel allelic mutation occurs where the G base is changed to an A base at the intron initiation site of the fifth intron, resulting in an alternative splicing mutation. Plants containing this mutant exhibit a dwarf phenotype, with a plant height approximately 40% of the wild parent. The seed setting rate is significantly increased compared to the wild parent, and the content of three active gibberellins, GA1, GA4, and GA7, in the stem is significantly reduced compared to the wild parent. This mutant is named... sdw1-2 ( Figure 9 These results indicate that Ft07.g34998 Genes do indeed have the function of regulating the height and seed setting rate of tartary buckwheat plants.

[0041] sdw1-2 The nucleotide sequence of the mutant is shown in SEQ ID NO.9, specifically: ATGTGTACTCAATCCTCCTCTATCACCCTCTCTCTTAGCTTTAACACATCATCTCTTCCACCATCGCCTAGTGTGCTATCAATTGTAGCAAGAGAGAAGCAATTTGGGCGTGGGTTGAGAAGATTGAGGTGTAATGCAACACCAAACCCCGACAACCCAAGTATCCAAGAGATCAAGAAAGAGGCATTTGGTGTACTTCAAAGTGATCGTATCCCACTTGTTAAGTGGCCTAATCATGATCATAATAAGGATAATTTGCAAATCCCAACTCATAATCTCAAGGGGAATGTATTGGACAAGAACAATGAACGAGTCGACGAGATTCGTTCAATCCTAAGATCGATGAATGACGGGGAAATATCGGTTTCTCCTTACGACACCGCATGGGTGGCCCTAGTGAAATACATAAATGGAAAACCACAGTTTTCTACTAGTTTGGAATGGATTGCAAAAAATCAACTTGAGGATGGGTCTTGGGGTGACAAAGACATGTTTGAAGCACACGATCGTATTATGAATACGTTAGCATGTGTAATTGCCCTTAGAACATGGAACATGTGTCTTAACCAATGCAACAAAGGGATGGAGTTTATAAGAGAAAATTTGAGCAAGCTTGAAGAAGAGAAAGATGAGCATATGCCAATCGGATTTGAGGTTGTTTTTCCTTCACTTATTGAGATTGCAAGAGATTTGGGGATTGAATTTTTGAATTCTGGATTCTTACAACACATTCATGCACAGAGAAACATAAAGCTCCAAAGGAAATATTGCACAACATACCAACATCATTGTTACACAGCCTAG; sdw1-2The amino acid sequence of the mutant-encoded sdw1-2 protein is shown in SEQ ID NO.10, specifically: MCTQSSSITLSLSFNTSSLPPSPSVLSIVAREKQFGRGLRRLRCNATPNPDNPSIQEIKKEAFGVLQSDRIPLVKWPNHDHNKDNLQIPTHNLKGNVLDKNNERVDEIRSILRSMNDGEISVSPYDTAWVALVKYINGKPQFSTSLEWIAKNQLEDGSWGDKDMFEAHDRIMNTLACVIALRTWNMCLNQCNKGMEFIRENLSKLEEEKDEHMPIGFEVVFPSLIEIARDLGIEFLNSGFLQHIHAQRNIKLQRKYCTTYQHHCYTA.

[0042] (4) Functional verification of transgenic Arabidopsis thaliana To further determine Ft07.g34998 The functions are described in this embodiment. Ft07.g34998 Genes (unmutated) and their role sdw1 The overexpression vector PBI121- of the mutant gene in the mutation SDW1 and PBI121- sdw1 The vector was constructed using PBI121 as the base vector, with SmaI and SacI restriction sites, and the inserted sequence fragments were as follows: SDW1 Gene (SEQ ID NO.1) and sdw1 Gene (SEQ ID NO.3). The PBI121-SDW1 and PBI121-sdw1 vectors were transformed into Agrobacterium using a freeze-thaw method. GV3101 In this study, Arabidopsis thaliana was genetically transformed using the flower-dipping method to obtain transgenic positive seedlings. Results showed that overexpression... Ft07.g34998 Genes and their role sdw1 The mutant gene (SEQ ID NO.3) in the mutant strain all increased the plant height of Arabidopsis thaliana, but overexpression of the mutant gene increased the plant height of Arabidopsis thaliana. sdw1 The plant height of the mutant gene was significantly lower than that of the overexpressed gene. Ft07.g34998 The plant height of the unmutated gene was significantly higher than that of the wild-type plant. Figure 10 ),show Ft07.g34998 It has the function of regulating plant height, and it is in sdw1 The mutation within the mutation did not lead to loss of function, but only weakened gene function; that is, when buckwheat contains... sdw1 When the SDW1 gene is present, the tartary buckwheat is semi-dwarf; when the SDW1 gene is present, it is tall.

[0043] 7. Molecular marker verification of the semi-dwarf, high-fruit-bearing gene sdw1 in tartary buckwheat and its application in high-yield, lodging-resistant breeding of tartary buckwheat. (1) Group building With semi-dwarf tall-stiff mutant sdw1 Using the female parent and the paternal parent, the buckwheat inbred line A323 (tall, with low seed setting rate and large grains, provided by the Buckwheat Germplasm Resource Bank of the College of Life Sciences, Guizhou Normal University) was used to obtain the F1 generation. The F1 generation was then self-pollinated to obtain the F2 generation, which consisted of 167 plants. The F2 generation showed segregation of plant height, with 118 tall plants and 49 semi-dwarf plants.

[0044] (2) Identification of genes for semi-dwarf and high-fruiting seedlings During the seedling stage, leaves from individual plants of the F2 population were collected, and leaf DNA was extracted using the CTAB method. Using the buckwheat leaf DNA as a template, quantitative real-time PCR amplification was performed using the designed SNaPshot-sdw1 primer pair. The amplification system for quantitative real-time PCR was as follows: 5 μL of 2×PARMS master mix, 0.4 μL of upstream universal primer F (10 μM), 0.15 μL of downstream primer R1 (10 μM), 0.15 μL of downstream primer R2 (10 μM), 100 ng of DNA template, and ddH2O to a final volume of 10 μL. The PCR amplification program was: 94℃ for 20 min; 94℃ for 20 s, 65-57℃ (-0.8℃ / cycle) for 1 min, cycle 10; 94℃ for 20 s, 57℃ for 1 min, cycle 32. Genotyping was performed based on the collected signals. The blue fluorescent signal near the X-axis represents the GG genotype, the green fluorescent signal near the Y-axis represents the AA genotype, and the green fluorescent signal in the middle represents the GA genotype.

[0045] (3) Field survey of plant height and fruit setting When the seed matures, for ( sdw1 The results of plant height and seed setting rate surveys for each individual plant in the F2 segregating population (mutant × A323) are shown in Table 3. Combined with genotyping results, it was found that in the F2 population, 118 tall plants belonged to both the GG and GA genotypes, with low seed setting rates (average stem height 125.42 cm, average seed setting rate 32.47%), while 49 short plants all belonged to the AA genotype, exhibiting semi-dwarfism and high seed setting (average stem height 79.63 cm, average seed setting rate 54.51%). Fortunately, this example yielded two semi-dwarf, high-seed, large-grained plants from the semi-dwarf, high-seed-setting individual plants. This demonstrates that the target fragment amplified by primer SNaPshot-sdw1 exhibits co-segregation of genotype and phenotype at its SNP sites. The sequence fragment shown in SEQ ID NO. 8 can serve as a molecular marker for identifying the semi-dwarf, high-seed-setting trait in buckwheat, and can be used for identifying true hybrids in hybrid offspring and for molecular marker breeding of high-yielding, lodging-resistant buckwheat.

[0046] Table 3 sdw1Genotype and plant height phenotype of individual plants in the F2 segregating population of mutant × A323 The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An sdw1 protein, characterized in that, The amino acid sequence of the sdw1 protein is shown in SEQ ID NO.4 or SEQ ID NO.

10.

2. A sdw1 gene encoding the sdw1 protein of claim 1, a semi-dwarf, high-fruiting buckwheat gene, characterized in that, The nucleotide sequence of the buckwheat semi-dwarf high-fruiting gene sdw1 is shown in SEQ ID NO.3 or SEQ ID NO.

9.

3. The application of the sdw1 protein of claim 1 or the sdw1 gene of semi-dwarf, high-fruiting buckwheat of claim 2 in the cultivation of semi-dwarf, high-fruiting buckwheat.

4. A molecular marker associated with buckwheat plant height and seed setting rate, characterized in that, The nucleotide sequence of the molecular marker is shown in SEQ ID NO.8, and a G / A mutation exists at the 61st base of the sequence.

5. A primer pair for amplifying the molecular marker of claim 4, characterized in that, The primer pairs include SNaPshot-sdw1 F with nucleotide sequences as shown in SEQ ID NO.5, SNaPshot-sdw1 R1 with nucleotide sequences as shown in SEQ ID NO.6, and SNaPshot-sdw1 R2 with nucleotide sequences as shown in SEQ ID NO.

7.

6. The molecular marker of claim 4 or the primer pair of claim 5 in the preparation of products for identifying buckwheat plant height and seed setting rate.

7. A product for identifying buckwheat plant height and seed setting rate, characterized in that, The product includes the primer pair as described in claim 5.

8. The use of the molecular marker of claim 4, the primer pair of claim 5, or the product of claim 7 in any of the following: (1) Identify the plant height and seed setting rate of buckwheat; (2) Screening for semi-dwarf, tall-fruiting bitter buckwheat; (3) Molecular breeding of buckwheat; (4) Improvement of buckwheat varieties.

9. A method for identifying the plant height and seed setting rate of tartary buckwheat, characterized in that, Includes the following steps: Using the genomic DNA of the buckwheat to be tested as a template, PCR amplification of the template was performed using the primer pair described in claim 5. The plant height and seed setting rate of the buckwheat to be tested were determined by the genotype of the PCR amplification results. If the genotype of the buckwheat to be tested is GG or GA, then the buckwheat to be tested is a tall-stalked buckwheat with a low seed setting rate; if the genotype of the buckwheat to be tested is AA, then the buckwheat to be tested is a semi-dwarf buckwheat with a high seed setting rate.

10. The application of reagents for editing the SDW1 gene in the cultivation of semi-dwarf, high-fruiting buckwheat, characterized in that... The nucleotide sequence of the SDW1 gene is as shown in SEQ ID NO.1.

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