Method for creating sorghum haploid and application thereof

Abstract

The application discloses a method for creating a sorghum haploid and application thereof and belongs to the field of biotechnology and agricultural biological breeding. The application creates a sorghum haploid inducer by knocking out or knocking down a SbTET11 gene by using a CRISPR / Cas9 system. The sorghum haploid inducer is crossed with a sorghum male sterile line as a male parent to obtain a hybrid offspring, i.e. a sorghum haploid. The application identifies and confirms that a sorghum gene SbTET11 has the ability to independently induce a haploid, provides a method for creating a sorghum haploid inducer, and converts SbTET11 into an explicit and implementable technical path for practical application, which provides an effective idea for promoting the breeding of sorghum and other species.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and agricultural bio-breeding, and in particular to a method for creating and applying haploid sorghum. Background Technology

[0002] Sorghum is the world's fifth most cultivated crop, with a planting area of ​​approximately 44.4 million hectares, accounting for 3.5% of total grain production. Due to its drought and heat tolerance, sorghum is well-suited for cultivation in regions where maize, rice, and wheat are difficult to grow, such as Africa, Central Asia, and central North America. Sorghum is not only an important food source in these regions but is also widely used in brewing (wine, vinegar), ethanol production, and silage, making it an ideal crop for addressing global climate change.

[0003] Traditional sorghum breeding faces challenges such as long cycles and high costs. Modern breeding technologies, such as double haploid (DH) technology, have been successfully applied in crops like maize due to their ability to rapidly fix recombinant haplotypes. However, the application of this technology in sorghum has lagged significantly. This lag is prominently reflected in the extremely low haploid induction rate (HIR): existing studies show that only two natural inducible lines (SMHI01 and SMHI021) were screened from 4000 sorghum germplasm accessions, with an induction efficiency of only 1-2%; the related patent (Patent, CN110546266A) from KWS also achieved an induction efficiency of only about 1%. Such low induction efficiency severely restricts the application of DH technology in sorghum breeding and makes it difficult to meet the needs of large-scale breeding. Therefore, developing new haploid inducible genes and improving the HIR of sorghum has become a key prerequisite for achieving efficient double haploid breeding in sorghum. There are two main approaches to achieving this goal: on the one hand, we can learn from the haploid-induced genes and mechanisms that have been discovered in crops such as maize; on the other hand, we need to focus on exploring and utilizing the haploid-induced gene resources of sorghum itself.

[0004] Drawing on experience from the development of maize haploid induction systems, scientists have identified several related genes in maize, including MTL / ZmPLA1 / NLD (hereinafter referred to as MTL), ZmDMP, ZmPLD3, ZmPOD65, and ZmGEX1. A single mutation of the major gene MTL in maize can achieve an induction rate of approximately 2-3%, while a double mutation of MTL with the minor gene ZmDMP produces a synergistic effect, significantly increasing the induction rate (HIR) to 8-15%. A superimposed mutation of MTL and ZmPLD3 results in an HIR of 1.19-4.13%, while the triple mutant of MTL, ZmDMP, and ZmPLD3 achieves an induction rate of 7-8%. Furthermore, a single mutation of ZmPOD65, encoding a sperm-specific peroxidase gene, can achieve a haploid induction rate of 1-7.7%. Mutations of ZmGEX1, a gene related to sperm and egg gamete expression, result in an average induction rate of 1.34%.

[0005] However, transferring the experience of maize DH breeding to other crops faces challenges: Research has found that the phospholipase A gene MTL is relatively conserved in monocotyledonous grasses, with orthologs existing. In rice (Oryzasativa), wheat (Triticum aestivum), and millet (Setaria italica), researchers have successively verified the ability of MTL homologs to induce haploids, achieving an induction rate of approximately 3%, through homologous gene cloning and gene editing. However, in dicotyledonous plants, due to the lack of orthologs of MTL, it remains unclear whether other phospholipase analogs participate in haploid induction. One research team, by further knocking out the OsDMP3 and OsDMP6 genes in rice, found that they neither possess independent haploid induction ability nor synergistically enhance the induction rate with the Osmtl mutation.

[0006] In dicotyledonous plants, the mutation effect of the MTL gene is not significant due to its poor conservation. In contrast, the DMP gene is conserved in both monocotyledonous and dicotyledonous plants, suggesting that mutations in DMP have great potential in developing haploid induction systems in dicotyledonous plants. In recent years, scientists have successively demonstrated the ability of the ZmDMP homologous gene to induce haploids in different species of dicotyledonous plants, including Arabidopsis thaliana, tobacco (Nicotiana tabacum), medicago truncatula, tomato (Solanum lycopersicon), cotton (Gossypium hirsutum), potato (Solanum tuberosum L.), cabbage (Brassica oleracea), rapeseed (Brassica napus), watermelon (Citrullus lanatus), soybean (Glycinemax), cucumber (Cucumis sativus L.), and pepper (Capsicum annuum L.), through bioinformatics analysis, homologous gene cloning, and gene editing. The HIR values ​​tested in these species were all less than 3% (the HIR of the zmdmp single mutant in maize was only 0.1-0.3%).

[0007] In summary, despite attempts in various crops, the 8% or higher maternal haploid induction efficiency required for commercial double haploid breeding has not yet been achieved in any crop other than maize. This is particularly evident in sorghum: a German patent (CN110546266A) mutated the homologous gene of sorghum MTL using the tilling method, achieving an induction efficiency of only about 1%, far below the threshold for industrial application. Therefore, developing relatively conserved haploid induction genes in both monocots and dicots is an effective approach to advancing DH breeding technology. Summary of the Invention

[0008] The purpose of this invention is to provide a method and application for creating sorghum haploids to solve the problems existing in the prior art. By using CRISPR / Cas9 technology to knock out the SbTET11 gene in sorghum to achieve haploid induction, a method for successfully creating sorghum haploid inducible lines and sorghum haploids has been developed. Furthermore, by using effective haploid selection markers, the induction frequency of haploid inducible lines has been evaluated, significantly promoting the progress of sorghum breeding.

[0009] To achieve the above objectives, the present invention provides the following solution:

[0010] This invention provides the application of SbTET11 inhibitors in inducing haploidization in sorghum, wherein the SbTET11 inhibitors include a CRISPR / Cas9 system that targets and knocks out or down the SbTET11 gene, and the nucleotide sequence of the SbTET11 gene is shown in SEQ ID NO.1.

[0011] The present invention also provides the application of SbTET11 inhibitors in the creation of haploid inducible lines, wherein the SbTET11 inhibitors include a CRISPR / Cas9 system that targets and knocks out or knocks down the SbTET11 gene, and the nucleotide sequence of the SbTET11 gene is shown in SEQ ID NO.1.

[0012] The present invention also provides the application of SbTET11 inhibitors in sorghum double haploid breeding, wherein the SbTET11 inhibitors include a CRISPR / Cas9 system that knocks out or knocks down the SbTET11 gene, and the nucleotide sequence of the SbTET11 gene is shown in SEQ ID NO.1.

[0013] Preferably, the CRISPR / Cas9 system includes sgRNA, the target sequence of which is the nucleotide sequence at positions 466-485 and 2475-2494 of the sequence shown in SEQ ID NO.1.

[0014] The present invention also provides a method for creating a sorghum haploid inducible line, comprising knocking out or knocking down the SbTET11 gene to obtain transgenic sorghum, which is a sorghum haploid inducible line; the nucleotide sequence of the SbTET11 gene is shown in SEQ ID NO.1.

[0015] Preferably, the SbTET11 gene is knocked out or knocked down using the CRISPR / Cas9 system.

[0016] Preferably, the CRISPR / Cas9 system includes sgRNA, the target sequence of which is the nucleotide sequence at positions 466-485 and 2475-2494 of the sequence shown in SEQ ID NO.1.

[0017] The present invention also provides a method for creating sorghum haploids, wherein the sorghum haploid induction line or its offspring prepared by the method is self-crossed or used as a male parent to cross with other wheat materials to obtain self-crossed offspring or hybrid offspring, which are the sorghum haploids.

[0018] The present invention also provides the application of the sorghum haploid induction line obtained by the above-described creation method in the preparation of sorghum haploids.

[0019] The present invention also provides the application of the sorghum haploid induction line obtained by the creation method or the sorghum haploid obtained by the creation method in sorghum double haploid breeding.

[0020] The present invention discloses the following technical effects:

[0021] 1) First discovery and functional confirmation of the SbTET11 gene: This invention is the first to identify and confirm that the sorghum gene SbTET11 has the ability to independently induce haploids. This solves the core problem of the long-standing lack of effective haploid inducing genes in the field of sorghum, achieving a breakthrough "from nothing to something".

[0022] 2) This invention proposes a specific method for creating haploid inducible lines of sorghum by knocking out or mutating the SbTET11 gene using gene editing technology (such as CRISPR-Cas9), and also provides a clear and feasible technical path for transforming the discovery of SbTET11 into practical applications.

[0023] 3) This invention utilizes sorghum material containing the GFP reporter gene as a recipient to analyze the haploid induction rate of plants after mutation of the SbTET11 gene. The results show that the induction rate is 0.451%. This is the first and only reported single-gene system that can achieve haploid induction in sorghum. This provides additional gene resources for the industrialization of sorghum double haploid breeding and also provides an application case for the development of double haploid breeding technology in other species (especially dicotyledonous plants).

[0024] 4) The use of sorghum haploid induction lines for double haploid breeding has greatly accelerated the breeding process. The induction line itself is a key technical point in the entire double haploid breeding process. The induction line itself is of great economic value and provides an effective approach for promoting double haploid breeding of sorghum and other species. Attached Figure Description

[0025] 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.

[0026] Figure 1 Comparison of TET11 amino acid sequences between different species;

[0027] Figure 2 Phylogenetic analysis of TET11 in different organisms;

[0028] Figure 3 This represents the tertiary structure of the SbTET11 protein.

[0029] Figure 4 The genome structure of SbTET11 and CRISPR / Cas9 target sites were obtained, along with information on mutant plants. Editing types of two homozygous frameshift mutant lines were obtained under two different background materials. Red letters represent PAM sites, bold black text represents 20nt target sequences, and yellow and blue box text represent the types of bases edited.

[0030] Figure 5 This is a schematic diagram for haploid identification in sorghum.

[0031] Figure 6 Phenotypic diagram of sorghum hybridization-induced haploid plants (Tx430-GFP genetic background);

[0032] Figure 7 This is a schematic diagram of the flow cytometry cell typing results. Detailed Implementation

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] Example 1: Obtaining the SbTET11 gene, a haploid-inducible gene in sorghum.

[0039] The genome sequence of the sorghum SbTET11 (Sobic.010G212700) gene, which was previously screened, is shown in SEQ ID NO.1, and its CDS sequence is shown in SEQ ID NO.2. The amino acid sequence of the protein encoded by the SbTET11 gene is shown in SEQ ID NO.3.

[0040] The amino acid sequences of tetraspanin (TET) proteins from sorghum (Sorghum bicolor), maize (Zea mays), rice (Oryza sativa), soybean (Glycine max), Arabidopsis thaliana, mouse (Mus musculus), and Homo sapiens (Homo sapiens) were compared for homology. Figure 1 As shown, the amino acid sequences of TET11 from sorghum, corn, rice, soybean, Arabidopsis, mouse, and Homo sapiens share 95%, 88%, 56%, 55%, 19%, and 17%, respectively. The amino acid sequences of the TET11 protein are extremely similar in the extracellular domain (EC2).

[0041] Multiple sequence alignments were performed on the SbTET11 gene of sorghum with the TET11 gene of other organisms. A phylogenetic tree was constructed in MEGA11.0 using the NJ method. The results are as follows: Figure 2 As shown in the figure, the TET11 gene is conserved in both monocot and dicotyledonous plants.

[0042] Example 2: Obtaining SbTET11 single-gene mutant lines of sorghum using CRISPR / Cas9 technology

[0043] Using the CRISPR / Cas9 gene editing tool, the SbTET11 gene in sorghum was knocked out under both the P898012-GFP and Tx430-GFP backgrounds (laboratory-created single-copy GFP lines, see invention patent CN120193001A), resulting in two knockout lines: P898012-GFP. tet11 Tx430-GFP tet11 The specific steps are as follows:

[0044] a) Selection of sgRNA target site sequences

[0045] The SbTET11 gene encodes a tetraspanin protein with zero alternatively spliced ​​transcripts. According to RNA-seq data, SbTET11 is specifically expressed in pollen, and it may be localized on the sperm cell membrane. Previous studies have shown that tetraspanins (TETs) are highly evolutionarily conserved transmembrane proteins in eukaryotic cells. TETs have four transmembrane domains, including a short extracellular circular domain (EC1), a very short intracellular circular domain (IC, typically containing only 4 amino acids), and a relatively long extracellular circular domain (EC2). The EC2 region is considered to contain most of the protein-protein interaction sites. The tertiary structure of the sorghum SbTET11 protein was predicted using the Swiss-Model online website (https: / / swissmodel.expasy.org / ), as shown in the figure. Figure 3 As shown. Since haploid induction may involve processes such as sperm-egg cell fusion, this invention designs two different sgRNA targets for the extracellular domain EC2 of SbTET11 to ensure complete knockout of the gene.

[0046] Two target sites, SbTET11-sgRNA-CR28 and SbTET11-sgRNA-CR8, were selected on the sorghum SbTET11 genome, located at the end of the first exon and the beginning of the second exon, respectively. The sequence of SbTET11-sgRNA-CR28 is CCTGCCGGAGACGACCCACC (SEQ ID NO.10), located at positions 466-485 on SEQ ID NO.1; the sequence of SbTET11-sgRNA-CR8 is GGATGCTGCAAGCCACCGAC (SEQ ID NO.11), located at positions 2475-2494 on SEQ ID NO.1. Specific gene structures and target site information are as follows: Figure 4 As shown, the amino acid sequence corresponding to the target site is as follows: Figure 1 As shown.

[0047] b) Construction of CRISPR / Cas9 vectors

[0048] The vectors CRISPR / SbTET11-sgRNA-CR28 and CRISPR / SbTET11-sgRNA-CR8 were obtained by constructing the target site DNA (20nt) sequences of SbTET11-sgRNA-CR28 and SbTET11-sgRNA-CR8 into the PCas9 vector, respectively (refer to Qu Lijia's Cell Research paper, which is publicly available and is a publicly disclosed vector).

[0049] c) Obtaining genetically modified sorghum

[0050] The CRISPR / SbTET11-sgRNA-CR28 and CRISPR / SbTET11-sgRNA-CR8 vectors obtained in step b) were transformed into Agrobacterium competent cells EHA105 (purchased from Shanghai Ziweidi Biotechnology Co., Ltd.); Agrobacterium containing CRISPR / SbTET11-sgRNA-CR28 and CRISPR / SbTET11-sgRNA-CR8 were transformed into sorghum P898012-GFP and Tx430-GFP recipients, respectively. After embryo transformation, screening, differentiation and rooting, T0 generation transgenic positive sorghum plants were obtained.

[0051] d) Identification of SbTET11 gene mutant strains in sorghum

[0052] DNA was extracted from the leaves of T0 generation positive plants in step c), and target site PCR amplification was performed using the following primers. The amplified products were sequenced, and the sequencing results were compared with the wild-type SbTET11 gene. The presence and type of SbTET11 mutations in different T0 generation transgenic sorghum lines were identified. The mutants were then planted in T1 generation plants to obtain stably inherited T2 seeds. The specific target information and editing types of the mutant lines subsequently used to evaluate haploid induction ability in this invention are shown in Table 1 and... Figure 4 As shown.

[0053] Table 1. Inducible line information under P898012-GFP and Tx430-GFP backgrounds.

[0054]

[0055] The sequence detection primers for the SbTET11 mutant (CR28 target site) were: tet-cr28-F: ATGGTGCGGTGCAGCAAC (SEQ ID NO.4) and tet-cr28-R: TGGATCGGGGAGAGGTTGC (SEQ ID NO.5). The PCR amplification product size was 503 bp. The sequencing primers were check-tet-cr28-F: CGCTCTTCCTCCTCATC (SEQ ID NO.6).

[0056] The primers for detecting the SbTET11 mutant (CR8 target site) sequence were: tet-cr8-F: AGCATGGCAACGTAGGG (SEQ ID NO.7) and tet-cr8-R: CATTTCCAGGCCGGGTAC (SEQ ID NO.8). The PCR amplification product size was 794 bp. The sequencing primers were check-tet-cr8-R: CAGTCGTTCTTCAGGTTG (SEQ ID NO.9).

[0057] Example 3: Method for producing haploids in sorghum

[0058] The four mutant lines and two chassis backgrounds obtained previously include: four SbTET11 single mutants (P898012-GFP). tet11-CR28 P898012-GFP tet11-CR8 and Tx430-GFP tet11-CR28 Tx430-GFP tet11-CR8 Two background plants, P898012-GFP and Tx430-GFP, were used as the chassis. The male-sterile sorghum line L407A, donated by Researcher Li Guiying of the Institute of Crop Science, Chinese Academy of Agricultural Sciences, was used as the female parent and is available to the public. These seven materials were used for field haploid induction ability assessment. The specific steps are as follows:

[0059] 1. Field hybridization experiment

[0060] Six materials used as induction lines were sown simultaneously, with the male-sterile line L407A sown in two additional stages to ensure its flowering coincided with that of the induction lines. Hybridization tests were conducted in Beijing (BJ) from May to September 2024 and in Hainan (HN) from February to April 2025. All materials were strictly banded after heading, and the six lines were used as pollen donors for hybridization with the male-sterile line L407A. After obtaining hybrids, the germination of the hybrid seeds was determined based on the presence or absence of GFP in the sorghum seed embryo and ploidy by flow cytometry of leaves. Specific results are shown in Tables 2 and 3.

[0061] Table 2. Assessment of haploid induction rate in sorghum (P898012-GFP background)

[0062]

[0063]

[0064] As shown in Table 2, after mutating the SbTET11 gene alone in the P898012-GFP background, the haploid induction rate was 0.499% (P898012-GFP). tet11 51 haploids were identified from 15,576 hybrids; as shown in Table 3, the haploid induction rate was 0.376% after mutating the SbTET11 gene alone in the Tx430-GFP background (Tx430-GFP). tet1135 haploids were identified from 12,479 hybrids. Wild-type P898012-GFP and Tx430-GFP were used as controls, and no haploids were found (a total of 5,413 hybrids were observed). The summarized data showed that the single mutation of SbTET11 in both genetic backgrounds could induce haploid production, with an average induction rate of 0.451%.

[0065] Table 3. Assessment of haploid induction rate in sorghum (Tx430-GFP background)

[0066]

[0067] 2. Identification of haploids in sorghum

[0068] In the inducible lines with backgrounds of P898012-GFP and Tx430-GFP, the plants contained a single copy of the GFP marker. When the paternal parent of the inducible line was crossed with the maternal parent of the sterile line, the embryos of the true hybrids carried the GFP marker from the paternal parent. Under ultraviolet light, the hybrid embryos showed green fluorescence, while the haploid embryos did not contain GFP fluorescence. Therefore, haploid and diploid embryos can be distinguished by fluorescence contrast. Specific GFP fluorescence and white light contrast images are shown below. Figure 5 As shown in the figure. Phenotypic diagram of sorghum hybrid-induced haploid plants. Figure 6 As shown.

[0069] 3. Flow cytometry verification of haploid phenotype

[0070] The leaves of the haploid plants shown in Tables 2 and 3 above were all subjected to flow cytometry ploidy identification. The specific method is as follows: The candidate haploids that germinated and were identified using GFP were sown in seed trays. The phenotype of individual plants was observed first: the diploid plants showed heterosis, with taller plants, wider leaves, and normal fertility, while the haploid plants after hybridization were shorter, with narrower leaves, more compact plant type, and sterility. Based on the above characteristics, potential haploids were judged a second time, and potential diploids that were judged incorrectly were removed. Young leaves were taken from potential haploid plants obtained after secondary screening, with leaves from diploid wild-type sorghum plants P898012-GFP or Tx430-GFP used as controls. Flow cytometry analysis was performed to identify haploid signal peaks. First, the nuclear signal of wild-type (diploid) cells was detected, and the peak position of the diploid cell nuclear signal was set at 42 (since the genetic material in diploid cells is twice that in haploid cells, the theoretical peak position of the haploid cell nuclear signal should be around 21). If the signal peak of the tested plant appeared around 42, it was considered to have the same enrichment position as the diploid cell nuclear signal, and the tested plant was considered diploid. If the nuclear signal peak of the tested plant appeared around 21, the tested plant was considered a haploid plant. Figure 7Flow cytometry has a smaller error rate, so it can accurately classify the tested plants into haploid and diploid groups after measurement.

[0071] Based on Examples 1-3, it can be concluded that the mutant sorghum SbTET11 gene can produce haploids. The average induction efficiencies of the induction lines with P898012-GFP and Tx430-GFP backgrounds were 0.499% and 0.376%, respectively, with an average induction efficiency of 0.451%. These results provide a potentially effective solution for the industrialization of double haploid breeding of sorghum and offer application examples for the development of double haploid breeding technology in other species.

[0072] The sequence involved in this invention:

[0073] SbTET11 gene sequence (SEQ ID NO.1, containing 2 exons):

[0074]

[0075] SbTET11 CDS sequence (SEQ ID NO.2):

[0076] ATGGTGCGGTGCAGCAACGGCCTGCTGGGCCTCCTGAACGCGGGGGTTCTGGTCCTCGCGGTCGTCGCGCTGGGGGGCGGCGCGTGGCTCAGCCACCGCGCGTCCACCACCGACTGCGAGCGGTTCCTGGAGCGGCCCGTCATCGCGCTGGGGGTGCTCCTCCTCGTGCTCTCCCTCGCGGGACTCGCGGGCGCGCTCTGCCGCGCCTCCTGCCTCCTCTGGCTCTACCTCCTCGCGCTCTTCCTCCTCATCCTGCTCCTCTTCGCCTTCACCGTCTTCGCCTTCGTCGTCACCAACCGCGGCGCCGGGTGGGTCGTCTCCGGCAGGGGGTACAAGGAGTACCGCCTTGGGGACTACTCCACCTGGCTGCAGAGGAGGGTCGAGAACTCGCAGAACTGGGCCAAGATCCGCAGCTGCCTCCAGGACGGCAAGGTGTGCGAGAAGCTCGCGGCCAGGAAGGAGACGGTCGCCCAGTTCGTCAACAGCAACCTCTCCCCGATCCAGTCTGGATGCTGCAAGCCACCGACAGGTTGCAACTTCACCTACCAGAGCGAGACTGTCTGGATCAAGCCCGCTGGCTTCAACACTACAAGTACAACTGACGACCCCGACTGCACCACATGGTCGAACGACCAGACCGTGCTCTGCTACGACTGCATGGCCTGCAAGGCAGGCGTGCTCGCCAACCTGAAGAACGACTGGAAGAAGATCGCCACCGTCAATATCATCTTCCTGATCTTCCTCATCGTCGTCTACTCCGTTGGGTGCTGCGCGTTCAGGAACAATCGGCAGGACAACTCGTACCCGGCCTGGAAATGA。

[0077] Amino acid sequence of SbTET11 protein (SEQ ID NO.3):

[0078] MVRCSNGLLGLLNAGVLVLAVVALGGGAWLSHRASTTDCERFLERPVIALGVLLLVLSLAGLAGALCRASCLLWLYLLALFLLILLLFAFTVFAFVVTNRGAGWVVSGRGYKEYRLGDYSTWLQRRVENSQNWAKIR SCLQDGKVCEKLAARKETVAQFVNSNLSPIQSGCCKPPTGCNFTYQSETVWIKPAGFNTTSTTDDDPDCTTWSNDQTVLCYDCMACKAGVLANLKNDWKKIATVNIIFLIFLIVVYSVGCCAFRNNRQDNSYPAWK*.

[0079] 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. The application of knocking out SbTET11 gene in creating sorghum haploid inducer line, characterized in that, The nucleotide sequence of the SbTET11 gene is shown as SEQ ID NO.

1.

2. Application of knocking out SbTET11 gene in highland brome double haploid breeding, characterized in that, The nucleotide sequence of the SbTET11 gene is shown as SEQ ID NO.

1.

3. The use according to claim 1 or claim 2, wherein the compound is of formula (I) ###0001### (I) or a pharmaceutically acceptable salt thereof. Knockout is performed by using a CRISPR / Cas9 system.

4. Use according to claim 3, wherein the compound is ###0002### The CRISPR / Cas9 system comprises sgRNA, and the target sequence of the sgRNA is the nucleotide sequence at positions 466-485 and 2475-2494 of the sequence shown as SEQ ID NO.

1.

5. A method of creating a sorghum haploid inducer line, characterized by, The transgenic sorghum is obtained by knocking out the SbTET11 gene, and is a haploid inducer of sorghum; the nucleotide sequence of the SbTET11 gene is shown as SEQ ID NO.

1.

6. The method of creating of claim 5, wherein, The SbTET11 gene is knocked out by using a CRISPR / Cas9 system.

7. The method of creating of claim 6, wherein, The CRISPR / Cas9 system comprises sgRNA, and the target sequence of the sgRNA is the nucleotide sequence at positions 466-485 and 2475-2494 of the sequence shown as SEQ ID NO.

1.

8. A method of creating a haploid of Sorghum bicolor, characterized by, The haploid inducer of sorghum prepared by the creation method of any one of claims 5-7 is crossed with other sorghum materials as a male parent to obtain hybrid offspring, which is the haploid of the sorghum.

9. The haploid inducer of sorghum prepared by the creation method of any one of claims 5-7 is used for preparing the haploid of sorghum.

10. The haploid inducer of sorghum prepared by the creation method of any one of claims 5-7 or the haploid of sorghum prepared by the creation method of claim 8 is used for breeding the double haploid of sorghum.

Images