Method for creating rice haploid inducer line with high efficiency of fixing variation and application thereof

Abstract

The application provides a method for creating a rice haploid inducer line capable of efficiently fixing variation and application thereof, and belongs to the field of agricultural technology. The application aims at the breeding demand of creating rice double haploid materials capable of efficiently fixing variation, identifies the function of key genes for inducing haploid in rice, screens out OsMATL and OsDMP genes with potential induction function, uses the genes as an inducer line of a male parent, and crosses the inducer line with a female parent with excellent traits, so that a haploid offspring carrying excellent traits of the female parent is obtained and chromosome doubling is performed, so that a rice excellent homozygous line is quickly obtained. The method provides a technical scheme for subsequent "one-stop" homozygosis of genetic mutant lines carrying excellent variation genes. The technical index in the application is clear, has strong operability, has certain technical innovation, and can provide technical system support for efficient fixation and utilization of excellent variation obtained by mutagenesis breeding.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of agriculture, and particularly relates to a method for creating a rice haploid induction line capable of efficiently fixing variation and application thereof. BACKGROUND

[0002] Traditional rice breeding methods require 6-8 generations of continuous selfing and selection to obtain a relatively stable line, which has a long cycle and is difficult to adapt to the current breeding situation of rapid iteration of varieties. Double haploid breeding (Double haploid, DH) is a breeding method that utilizes natural or artificial haploid induction (Haploid induction, HI) and then obtains diploids through chromosome doubling, which can obtain a pure line in only two generations, greatly shortening the breeding process and reducing the investment of manpower and material resources.

[0003] The MATL (MATRILINEAL) gene is a key gene derived from corn material Stock6 that controls the haploid induction trait of corn. The gene encodes a phospholipase that is specifically expressed in sperm cells and is involved in the metabolic pathway of phospholipid degradation and linolenic acid production. The in vivo haploid induction function mediated by the MATL gene is relatively conservative in monocotyledonous plants. OsMATL mutants have been successfully created in rice, with a seed setting rate of 2-15% and different types of aborted grains. Another key gene that controls haploid induction trait is ZmDMP, which encodes a DMF679 domain membrane protein that is specifically expressed in pollen. On the basis of MATL mutation, ZmDMP knockout can increase the haploid induction rate (Haploid induction rate, HIR) by 5-6 times, significantly improving HIR.

[0004] Currently, a method has been realized in corn for hybridizing haploid induction lines with edited specific gene functions with excellent parents to obtain homozygous offspring lines with specific gene editing. For example, this technology is used to quickly obtain homozygous mutant plants of leaf opening angle gene ZmLG1, realizing efficient homozygosis and fixation of excellent variation.

[0005] However, there is no method for quickly obtaining homozygous lines of excellent variation in rice by using haploid induction trait related genes. SUMMARY

[0006] To solve the above technical problems, the application provides a method for creating a rice haploid induction line capable of efficiently fixing variation and application thereof. The application aims at the breeding demand of creating a rice double haploid material capable of efficiently fixing variation, identifies the function of key genes for inducing haploid in rice, screens out the OsMATL and OsDMP genes with potential induction function, uses the genes as the male parent of the induction line, and crosses the induction line with the female parent with excellent traits to obtain haploid offspring carrying excellent traits of the female parent and to double the chromosomes to quickly obtain a rice excellent homozygous line. The method provides a technical solution for the "one-stop" homozygosis of the genetic mutant line carrying excellent variation genes. The technical index in the application is clear, has strong operability, has certain technical innovation, and can provide technical system support for efficiently fixing and utilizing excellent variation obtained by mutagenesis breeding.

[0007] To achieve the above purpose, the application adopts the following technical solution:

[0008] The application provides a method for creating a rice haploid induction line capable of efficiently fixing variation, comprising the following steps:

[0009] (1) Identifying haploid induction effect related genes:

[0010] a. predicting the structure of the haploid induction effect gene in rice; b. predicting the orthologous genes of the haploid induction effect gene in rice; c. detecting the expression pattern of the haploid induction effect related genes; d. performing subcellular localization of the haploid induction effect related genes;

[0011] (2) Creating and identifying mutants with haploid induction effect:

[0012] a. creating mutants:

[0013] In combination with the structural characteristics of the haploid induction effect gene in rice, a mutation site is designed at an active site to destroy the protein function, and a knock-out line is created in the background material of indica rice variety Huahang 48, and the created knock-out line includes single gene mutants and double gene mutants;

[0014] b. identifying positive mutant lines:

[0015] The mutant line is self-pollinated to the T2 generation, RNA extraction and qRT-RCR analysis are performed, the gene sequence detected by qRT-RCR is subjected to Sanger sequencing, and is compared with the gene sequence of the wild type of Huahang 48, and in combination with the qRT-RCR and Sanger sequencing results, if the gene expression amount is down-regulated and the gene sequence is mutated as expected, the positive mutant line can be determined;

[0016] (3) Identifying related characteristics of the positive mutant line:

[0017] Positive mutant lines were self-pollinated, and putative haploids were identified by comparing plant height and leaf characteristics with diploid siblings and wild type (WT) Huanghai 48 in the progeny, and the ploidy of putative haploids was confirmed by flow cytometry analysis, and the self-pollination HIR was determined, HIR (%)=(number of haploids / total number of plants) x 100%, the self-pollination seed setting rate (SSR) was determined, SSR (%)=(number of filled seeds / (number of filled seeds+number of empty seeds)) x 100%, and Duncan's new multiple range test was used to analyze the significant difference in mean value of seed setting rate between multiple groups of samples;

[0018] (4) Identification of haploid induction ability of positive mutant lines:

[0019] Positive mutant lines of T2 generation were self-pollinated and used as male parents, and photoperiod and temperature sensitive sterile indica rice line Hang 93S was used as female parent to evaluate HIR, and Huanghai 48 was used as a control male parent, and HIR and SSR were determined after seed harvest.

[0020] Preferably, the prediction method of the rice haploid induction effect gene structure is as follows: the protein domain of the haploid induction effect related gene is predicted by using Pfam and UniProt websites, and the motif structure, affinity base active site and proton acceptor active site key structures contained in the gene are focused on.

[0021] Preferably, the prediction method of the rice haploid induction effect gene orthologous gene is as follows: the full-length amino acid sequence of the identified haploid induction effect gene of other plants is used for BLASTP, and the sequence database of rice is searched, genes with amino acid sequence identity >40% are selected and a phylogenetic tree is constructed, and the homologous relationship of the selected genes with the identified haploid induction effect genes of other plants is analyzed.

[0022] Preferably, the detection method of the expression pattern of the haploid induction effect related gene is as follows: total RNA is extracted from indica rice Huanghai 48 plants for mRNA reverse transcription, and qRT-PCR is performed on the knocked-out gene, OsUBQ is used as a reference gene, each sample has 3 biological replicates, each replicate includes 3 technical replicates, and the data is processed by 2 －△△Ct method to detect the expression pattern of the haploid induction effect related gene.

[0023] Preferably, the subcellular localization method of the haploid induction effect related gene is: cloning the protein coding sequence without stop codon into the SpeI-BamHI site of PAN580-GFP vector, co-transforming the recombinant plasmid with mCherry into the protoplast of rice, using the vector without the target gene for control experiment, using the endoplasmic reticulum localized mCherry as the plasma membrane marker, using the nucleus localized mCherry as the nucleus marker, using the vesicle localized mCherry as the vesicle marker, using FM4-64 as the cell membrane dye, capturing the green fluorescence signal and the red fluorescence signal after incubation at 28°C for 12-16 hours.

[0024] Preferably, the gene used in the method for creating single gene mutant is OsMATL or OsDMP, and the genes used in the method for creating double gene mutant are OsMATL and OsDMP.

[0025] Further preferably, the OsDMP is any one of OsDMP1 (LOC_Os08g01530), OsDMP2 (LOC_Os05g48840), OsDMP3 (LOC_Os01g29240), OsDMP4 (LOC_Os02g27800) and OsDMP5 (LOC_Os12g22270).

[0026] Still further preferably, the genes used in the method for creating double gene mutant are OsMATL and OsDMP1, or OsMATL and OsDMP3.

[0027] The detailed steps of the above method are as follows:

[0028] (1) Identifying haploid induction effect related genes:

[0029] a. Predicting the structure of rice haploid induction effect genes: using the website Pfam (http: / / pfam.xfam.org / ) and UniProt (https: / / www.uniprot.org / ) to predict the protein domain of haploid induction effect related genes, focusing on key structures such as motif structure, affinity base active site and proton acceptor active site contained in the gene.

[0030] b. Predicting orthologs of haploid induction effector genes in rice: Full-length amino acid sequences of haploid induction effector genes identified in other plants were used for BLASTP to search the sequence database of rice. Genes with amino acid sequence identity > 40% were selected to construct a phylogenetic tree. The full-length candidate genes of rice and haploid induction effector genes of other plants were aligned by MUSCLE embedded in software MEGA11 using the maximum likelihood method, with a Bootstrap value of 1000, and a phylogenetic tree was constructed using the amino acid sequences of the above genes.

[0031] c. Detecting the expression pattern of haploid induction effector-related genes: RNA extraction and qRT-RCR analysis were performed to determine the expression of haploid induction effector-related genes in the background material. Total RNA was extracted from the roots, stems, leaves, leaf sheaths, ligules, young panicles, immature anthers, mature anthers, and 35-day seeds of the indica rice "Huahang 48" plant using an RNA plant extraction kit (Vazyme, vazyme.com).

[0032] Evo M-MLV reverse transcription premix kit (AG, https: / / agbio.com.cn / ) was used for mRNA reverse transcription. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed on the knocked-out genes using a PerfectStart Green qPCR SuperMix kit (Vazyme, vazyme.com) and a StepOnePlus fluorescent quantitative PCR instrument (Applied Biosystems, Thermo Fisher Scientific-CN), with OsUBQ as the reference gene. Each sample had 3 biological replicates, each replicate included 3 technical replicates, and the data was processed using the 2 －△△Ct method.

[0033] d. Subcellular localization of haploid induction effector-related genes: Subcellular localization helps to further verify the haploid induction effect of candidate genes. The protein coding sequence without a stop codon was cloned into the SpeI-BamHI site of the PAN580-GFP vector. The recombinant plasmid was co-transformed with mCherry fusion into protoplasts of rice. A control experiment was performed using a vector without the target gene. Endoplasmic reticulum-localized mCherry was used as a plasma membrane marker, nucleus-localized mCherry was used as a nuclear marker, vesicle-localized mCherry was used as a vesicle marker, and FM4-64 was a cell membrane dye. After incubation at 28°C for 12-16 hours, green and red fluorescent signals were captured by a laser scanning confocal microscope (Zeiss / LSM800, https: / / www.zeiss.com).

[0034] (2) Creation and identification of mutants with haploid induction effect:

[0035] a. Creation of mutants: Based on the structural characteristics of the predicted rice haploid induction effect genes, mutation sites were designed at the active sites to disrupt protein function. The indica rice variety Huahang 48 was used as the background material to create knock-out lines. Rice plants were grown in a phytotron during the normal growing season in the local area, with an environmental temperature of about 28℃ and a photoperiod of 16 hours of light / 8 hours of darkness. The created knock-out lines included single gene mutants (single mutants) and double gene mutants (double mutants). Genetic transformation of rice was completed by the company Biorun Bioscience Co., Ltd (biorun.com).

[0036] b. Identification of positive mutant lines: The mutant lines were self-pollinated to the T2 generation, and the T2 plants were subjected to RNA extraction and qRT-RCR analysis using the method in (1). The gene sequences detected by qRT-RCR were sequenced by Sanger sequencing and compared with the gene sequences of the wild type Huahang 48. Based on the qRT-RCR and Sanger sequencing results, if the gene expression is significantly down-regulated and the gene sequence has the expected mutation, it can be determined as a positive mutant line.

[0037] (3) Identification of related characteristics of positive mutant lines (induction lines): The positive mutant lines were self-pollinated, and the putative haploids were identified by comparing the plant height and leaf characteristics of the offspring with those of the diploid siblings and the wild type Huahang 48 (WT). Flow cytometry analysis was used to confirm the ploidy of the putative haploids, and the HIR of selfing was determined. HIR (%) = (number of haploids / total number of plants) x 100%. The seed setting rate (SSR) of selfing was also determined, SSR (%) = (number of filled seeds / (number of filled seeds + number of empty seeds)) x 100%. Duncan's multiple-range test was used to analyze the significant differences in mean values of the seed setting rates between multiple groups of samples.

[0038] (4) Identification of haploid induction ability of positive mutant lines (induction lines): The positive mutant lines (self-pollinated to the T2 generation) were used as the male parent, and the indica rice line Hanguang 93S, which is a photoperiod-sensitive sterile line, was used as the female parent for hybridization to evaluate the HIR at hybridization. Huahang 48 was used as the control male parent. The growth conditions of the hybridization parents and offspring were the same as those of Huahang 48, which was used as the background material for the knock-out lines. The seeds were harvested about 30 days after pollination (DAP). The methods for determining HIR and SSR were the same as those used for the identification of positive mutant lines.

[0039] The present application also provides an application of the method in rice breeding.

[0040] The application also provides application of the rice haploid inducer line selected by the method in rice breeding.

[0041] The application has the following beneficial effects:

[0042] 1. The innovation of the application mainly includes: further identifying and clarifying the functions of the key haploid induction genes OsMATL and OsDMP in rice, providing a technical solution for identifying other haploid induction genes in rice; successfully creating a single-gene mutant matl and a double-gene mutant matl dmp with haploid induction effect, wherein the haploid induction rate of matl dmp is higher than that of matl, thereby widening the method of creating haploid induction lines to double genes and widening the approach of creating rice haploid induction lines; through crossbreeding of the induction line and an excellent parent, the potential of the double mutant for efficient fixation of variation in outcrossing is further identified and confirmed, thereby providing successful experience for the application of haploid induction lines in breeding practice.

[0043] The application has clear technical indicators, strong operability and certain technical innovation, and provides induction materials and technical systems for efficient fixation and utilization of high-quality variation.

[0044] 2. The application has a complementary effect on various technical defects in the prior art:

[0045] (1) In the previous research, the research on identifying haploid induction related genes and creating haploid induction lines is mostly concentrated in corn and other crops, and there is less research in rice. In corn, the creation of haploid induction lines and the efficient fixation of excellent variation have made rapid progress, for example, double-gene mutation of ZmMATL and ZmDMP can increase the haploid induction rate (HIR) of corn by 5-6 times (Zhong et al., 2019), and crossbreeding of a corn haploid induction line with the function of editing specific genes with an excellent female parent can quickly obtain a homozygous mutant plant of the leaf opening angle gene ZmLG1 (Wang et al., 2019). The current research on MATL genes in rice is limited to the effect of OsMATL mutation on the seed setting rate (SSR) of rice (Wen et al., 2021), and there is less mature haploid induction line created and used for fixation of variation.

[0046] The application aims at creating a rice haploid induction line with efficient fixation of variation on rice, which aims to create a rice haploid induction line with high efficient fixation of excellent variation potential, to fill the gap of related research on rice, and to provide technical support for efficient homozygous excellent variation. The application breaks through the previous limitation of shallow research on haploid induction genes, combines gene function identification, gene editing and "induction line x excellent strain" hybridization technology, and preliminarily obtains multiple rice haploid induction lines with high efficient variation potential and their creation methods. The method is a relatively complete method for creating a rice haploid induction line with efficient fixation of variation on rice, and has strong innovation significance.

[0047] (2) The previous research only carried out simple function research on single gene, created single gene mutation induction line, such as only through gene structure analysis and expression detection to identify OsMATL gene, and preliminarily explored the seed setting rate of the mutant of the gene (Wen et al., 2021); there are also researches trying to create single gene mutation induction line of OsDMP but failed (Liu et al., 2024).

[0048] The application combines gene structure prediction and expression detection, protein subcellular localization and other technologies, and identifies the functions of two key genes OsMATL and OsDMP for inducing haploid in rice, making the function identification of haploid induction genes more accurate; OsMATL and OsDMP double gene mutants are created as haploid induction lines, and it is found that the double gene mutants of OsMATL and multiple OsDMP family genes have high haploid induction rate, such as matldmp1 (8.6%) and matldmp3 (6.05%), which are higher than matl (5%), which widens the range of haploid induction candidate genes and provides the research idea of creating "double gene mutation induction line".

[0049] (3) The previous research is difficult to consider the seed setting rate (SSR) and the induction rate (HIR) of haploid induction line, and the two even have antagonistic relationship, while in fact, only the induction line with both of them has strong production application potential. Some researches only focus on SSR, and do not systematically statistics the haploid HIR (Wen et al., 2021); some researches mutate haploid induction genes, and the SSR of the mutants is low (less than 40%), which is not conducive to obtain haploid offspring (Liu et al., 2024).

[0050] The application takes into account the SSR and HIR of the haploid induction line, and a haploid induction line with high SSR and HIR is obtained, in particular, a double gene mutation induction line with high SSR is obtained. For example, the average SSR of the fourth exon mutant (M4-1) of OsMATL is nearly 50%, and the SSR of the matl dmp1 (D1-MD-1) and matl dmp4 (D4-MD-1) double gene mutation induction lines is close to that of M4-1, which shows that the double gene mutation induction line obtained by the application has higher HIR than the single gene mutation induction line, and the SSR is not reduced due to the increase of the mutant gene. The double gene mutation induction line obtained by the application has high SSR and HIR, and has great application potential in haploid breeding and rice production.

[0051] (4) The previous research has not verified the induction ability of the induction line in breeding by crossing the haploid induction line with the high-quality female parent, and the haploidization and excellent variation fixation ability of the high-quality parent have not been verified (Wen Q et al., 2021).

[0052] In the application, the matl dmp of different mutation types is used as the male parent to cross with Hui 93S (the female parent), and haploids are detected in the offspring of most cross combinations, which shows that the double gene mutation induction line of OsMATL and OsDMP has the potential to quickly haploidize and fix the excellent variation after crossing with the female parent carrying the excellent variation. The application provides more high-quality haploid induction lines for the creation of rice haploids.

[0053] (5) The application first finds that the mutation of the DGA / G motif and the proton acceptor active site of the third exon of the OsMATL gene can induce high-frequency haploids (HIR reaches 5.78%), which provides a candidate site for creating more haploid induction lines and enriching the rice haploid induction line library material by using the OsMATL gene. BRIEF DESCRIPTION OF DRAWINGS

[0054] Figure 1 It is the phylogenetic tree of OsDMP constructed by the adjacency method using the MEGAX program in Example 1 of the application, wherein the numbers beside the branches represent the percentage of bootstrap values of 1000 replicates, and the scale represents the evolutionary distance of 0.1 amino acid residues at each position in the sequence;

[0055] Figure 2 It is the expression amount of OsDMP and OsMATL in Example 2 of the application, using root, stem leaf, leaf sheath, ligule, young ear, mature anther and seed samples, three biological and technical repeats were performed, and these values are expressed in the form of average value ± standard error (SE);

[0056] Figure 3Figure 2 shows subcellular localization of OsMATL and OsDMP in Example 2 of the present application, Bar = 2 μm;

[0057] Figure 4 Figure 3 shows OsMATL gene structure and knockout target sequence in Example 3 of the present application, wherein: (A) OsMATL gene structure, each part represents an exon, from left to right, the first to the fourth exon; (B) OsMATL knockout target sequence, S1 is the first exon knockout target, S2 is the third exon knockout target, S3 is the fourth exon knockout target;

[0058] Figure 5 Figure 4 shows OsMATL fourth exon mutant and target and mutation type of double knockout lines produced by OsMATL and OsDMP1, OsDMP3, OsDMP4, and OsDMP5, respectively, in Example 4 of the present application;

[0059] Figure 6 Figure 5 shows the synergistic effect of OsMATL and OsDMP mutations on HI in Example 4 of the present application, where D is diploid and H is haploid; (A) Huaxiang 48 and various knockout combinations, Bar = 5 cm; (B) SSR of selfing of Huaxiang 48 and various mutant derivatives, values are mean ± standard deviation, ****, P < 0.0001; ns, not significant (two-tailed Student's t-test); (C) selfing HIR in matl, matl dmp1, and matl dmp3 mutant lines; (D) plant type of diploid and haploid selfing, Bar = 8 cm; (E) ear morphology of diploid and haploid, Bar = 2 cm; (F) cell ploidy level determined using flow cytometry; (G) grain shape of diploid and haploid, Bar = 5 mm; (H) plant type of hybrid diploid and haploid, Bar = 14 cm. DETAILED DESCRIPTION

[0060] The following examples are intended to illustrate but not limit the scope of the present application. Modifications or substitutions of the method, steps, or conditions of the present application, without departing from the spirit and essence of the present application, are within the scope of the present application. The products, reagents, instruments, and equipment used in the following examples are commercially available, and the methods used are consistent with the commonly used methods, unless otherwise specified.

[0061] The technical solutions of the present application are further described in detail below in conjunction with the examples.

[0062] Example 1 Identification of OsDMP candidate genes in rice

[0063] OsDMP is a key gene in rice that controls haploid induction traits. Five ZmDMP-like proteins were identified in the rice genome, with a sequence identity of >40%. According to the similarity with ZmDMP from large to small, they are named OsDMP1 (LOC_Os08g01530), OsDMP2 (LOC_Os05g48840), OsDMP3 (LOC_Os01g29240), OsDMP4 (LOC_Os02g27800) and OsDMP5 (LOC_Os12g22270), and the sequence similarity of these proteins is much higher than that of other proteins. Phylogenetic analysis shows that OsDMP1, OsDMP2, OsDMP3, OsDMP4 and OsDMP5 are closely related to ZmDMP orthologs, and have proven HI ability in corn, Arabidopsis, tomato, Medicago truncatula and tobacco Figure 1 It is reported that sequence similarity greater than 40% is considered to have the same function, therefore OsDMP1, OsDMP2, OsDMP3, OsDMP4 and OsDMP5 are selected as candidate genes for haploid induction.

[0064] OsDMP1 (LOC_Os08g01530)

[0065] https: / / www.ricedata.cn / gene / gene_info.aspx?id=LOC_Os08g01530

[0066] OsDMP2 (LOC_Os05g48840)

[0067] https: / / www.ricedata.cn / gene / gene_info.aspx?id=LOC_Os05g48840

[0068] OsDMP3 (LOC_Os01g29240)

[0069] https: / / www.ricedata.cn / gene / gene_info.aspx?id=LOC_Os01g29240

[0070] OsDMP4 (LOC_Os02g27800)

[0071] https: / / www.ricedata.cn / gene / gene_info.aspx?id=LOC_Os02g27800

[0072] OsDMP5 (LOC_Os12g22270)

[0073] https: / / www.ricedata.cn / gene / gene_info.aspx?id=LOC_Os12g22270

[0074] Example 2 Analysis of expression patterns of haploid induction related genes in rice

[0075] The expression patterns of OsMATL and OsDMP were analyzed by qRT-PCR and subcellular localization experiments. The reference for the sequence of OsMATL is: Wen Q, Jia S, Wang JF, et al. Creation and analysis of a mutant of the rice haploid induction gene OsMATL [J]. Crop Science, 2021, 5: 827-836.

[0076] Studies have shown that the expression levels of haploid induction related genes MATL and DMP are high in mature anthers. To explore the tissue expression patterns of OsMATL, OsDMP1, OsDMP3, OsDMP4 and OsDMP5 genes, qRT-PCR experiments showed that OsMATL, OsDMP1, OsDMP3 and OsDMP4 were highly expressed in mature anthers, and the expression levels in other tissues were very low; OsDMP5 had the highest expression in young panicles, and had a small amount of expression in leaves and mature anthers, etc. Figure 2 Subcellular localization experiments of rice protoplasts confirmed the subcellular localization of these candidate genes, and found that OsMATL was localized in the cell membrane, OsDMP1 was localized in the endoplasmic reticulum, OsDMP3 was localized in the vesicle, OsDMP4 was localized in the endoplasmic reticulum, and OsDMP5 was localized in the endoplasmic reticulum and nucleus. Figure 3

[0077] The results of expression pattern analysis showed that the expression patterns and subcellular localizations of OsMATL, OsDMP1, OsDMP3, OsDMP4 and OsDMP5 were similar to or related to ZmPLA1 / MATL / NLD and ZmDMP. Therefore, these findings mean that OsMATL, OsDMP1, OsDMP3, OsDMP4 and OsDMP5 may have haploid induction function in rice (dmp2 mutant did not receive seeds, so the tissue expression pattern was not studied, see Example 4 for details).

[0078] Example 3 Creation and identification of OsMATL mutants with haploid induction effect

[0079] a. Create OsMATL gene mutants at different active sites.

[0080] ​OsMATL is a key gene in rice that induces haploid traits. Using the websites Pfam (http: / / pfam.xfam.org / ) and UniProt (https: / / www.uniprot.org / ), the protein domains of the OsMATL (LOC_Os03g27610) gene were predicted. It was found that this gene also contains three motifs (GXGXXG, GXSXG, and DGA / G), one affinity group active site, one proton acceptor active site, and one S-palmitoylation or S-farnesylation site. Figure 4 A).

[0081] Knockout lines were created using the indica rice variety Huahang 48. The genetic transformation of the rice was carried out by Biorun Bioscience Co., Ltd. To obtain mutants at different OsMATL loci, three single-target sites were designed in the coding region. Figure 4 B) to generate frameshift mutations at different sites. S1 is designed on exon 1 to completely disrupt the function of the MATL protein; S2 is designed on exon 3 at the DGA / G motif and proton acceptor active site; S3 is designed on exon 4 8 bp upstream of the natural mutation site in maize.

[0082] b. Identify the haploid induction effect of mutants at different active sites of the OsMATL gene.

[0083] Mutants at different sites of OsMATL were identified in the T2 generation of mutants. These mutants carried base insertions or deletions and did not contain CRISPR / Cas9 transgenic elements. The results showed that the proton active site of the third exon of OsMATL is the most suitable site for inducing haploids in indica rice, specifically identified by morphological characteristics, seed setting rate (SSR), and haploid induction rate (HIR).

[0084] In terms of morphological characteristics, no significant morphological differences were observed between any mutant and the corresponding wild type. However, haploid offspring obtained through self-pollination exhibited typical haploid characteristics such as reduced body size and male sterility. The estimated ploidy level of the haploids was also confirmed by flow cytometry. Regarding SSR (Self-Rating Induction), the SSR of the OsMATL coding region mutants ranged from 20.3% to 22.9%, all significantly lower than the wild type. There were no significant differences in SSR among mutants at the three different mutation sites. Regarding HIR (Self-Rating Induction), most OsMATL mutants were capable of inducing haploidy during self-pollination, with an HIR of 3.9% to 11.5%. Among the three mutation sites, the S2 site mutant had the highest HIR at 7.8%, with the S2 site 5bp deletion mutant showing a haploid induction rate as high as 11.5%. The S3 site mutant had the second highest HIR at 5.8%, while the S1 site mutant had the lowest at 4.7%.

[0085] Example 4 Creation and identification of OsMATL and OsDMP double mutants capable of efficiently fixing variation

[0086] a. Creation of double mutants.

[0087] According to the results of previous studies, double knockout lines of OsMATL and OsDMP were created by knocking out the fourth exon of OsMATL and OsDMP1, OsDMP2, OsDMP3, OsDMP4, and OsDMP5, respectively, in Huahang 48. Meanwhile, mutants of the fourth exon of OsMATL were created as controls. The genetic transformation of rice was completed by Biorun Bioscience Co., Ltd.

[0088] The target points and mutation types of the fourth exon of OsMATL mutants, and double knockout lines of OsMATL and OsDMP1, OsDMP3, OsDMP4, and OsDMP5 are shown in Table 1. Figure 5 The "Lines" column, WT, the wild-type material of Huahang 48 without mutation, is used as a background control; HH48 matl4-1 , single mutation of the fourth exon of OsMATL; HH48 matl4-ndmp1-n (n is a number, and different n represents different mutation types. The same below), double mutation of OsMATL and OsDMP1; HH48 matl4-ndmp3-n , double mutation of OsMATL and OsDMP3; HH48 matl4-1dmp4-1 , double mutation of OsMATL and OsDMP4; HH48 matl4-ndmp5-n , double mutation of OsMATL and OsDMP5. The "Mutation types" column, + is base insertion, and the red base is the inserted base, and - is base deletion.

[0089] b. Identification of the haploid induction effect of double mutants.

[0090] In the T2 generation, double mutants of OsMATL and OsDMP1, OsDMP3, OsDMP4, and OsDMP5 (denoted as matldmp1, matldmp3, matldmp4, and matldmp5) and the fourth exon single mutant of OsMATL (denoted as matl) were obtained, which carried base insertion or deletion and had no CRISPR / Cas9 transgene elements.

[0091] The identification results showed that OsDMP1 and OsDMP3 in indica rice exhibited synergistic haploid activity with OsMATL. Specifically, the double mutants were identified based on morphological characteristics, seed setting rate (SSR), and haploid induction rate (HIR). Regarding morphological characteristics, no significant morphological differences were observed between most mutants and the corresponding wild type (Huahang 48). Figure 6 A), but the haploid offspring obtained through self-pollination exhibit typical haploid characteristics such as reduced body size and male infertility. Figure 6 D, 6E), the estimated ploidy level of haploids was also confirmed by flow cytometry. Figure 6 F). Regarding SSR, the SSR rates for wild-type and matl were 92.5% and 49.8%, respectively. Significant differences were found in the SSR rates of matl dmp1, matl dmp3, and matl dmp5 compared to matl, indicating that mutations in OsDMP1, OsDMP3, and OsDMP5 can reduce SSR. Figure 6 B). Regarding HIR, matl had an HIR of 1.9% (number of haploids / total number of progeny = 1 / 52, the same below), matldmp1 and matldmp3 had HIRs of 2.2% (8 / 363) and 2.7% (5 / 188) respectively. Figure 6 C).

[0092] c. Identify the potential of double mutants to efficiently fix variations in crossbreeding.

[0093] Cross-pollination experiments showed that when all mutants with different mutation types were used as male parents and crossed with Hang93S, haploids were detected in all mutants except for D1-MD-3. Haploids were also detected in all other combinations of different mutation types. Figure 6 The OsMATL and OsDMP double mutants (matldmp1 and matldmp3) were tested for HIR (8.6% and 6.05%, respectively), while no haploid individuals were detected in any of the individuals obtained by pollination with the wild-type (Huahang 48) paternal parent. All haploids were examined by flow cytometry. The HIR of double mutant crosses (matldmp1 and matldmp3, with average HIR of 8.6% and 6.05%, respectively) was higher than that of single mutants (matl, with an average HIR of 5%). These results indicate that the OsMATL and OsDMP double mutants (matldmp1 and matldmp3) have the potential to rapidly haploidize and fix the superior variants after crossing with maternal parents carrying superior variants.

[0094] 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. A method for creating a rice haploid inducer line with high efficiency of fixing variation, characterized in that, Comprising the following steps: (1) Identifying haploid induction effect related genes: a. Predicting the structure of rice haploid induction effect genes; b. Predicting the orthologous genes of haploid induction effect genes in rice; c. Detecting the expression pattern of haploid induction effect related genes; d. Subcellular localization of haploid induction effect related genes; (2) Creating and identifying mutants with haploid induction effect: a. Create mutants: In combination with the structural characteristics of the haploid induction effect gene of rice, a mutation site is designed at an active site to destroy the protein function, and a knock-out line is created by taking the indica rice variety Huahang 48 as background material. The created knock-out line includes single gene mutants and double gene mutants; the gene used for creating the single gene mutant is OsMATL or OsDMP , the gene used for creating the double gene mutant is OsMATL and OsDMP ; the OsDMP is any one of OsDMP1 ( LOC_Os08g01530 ), OsDMP2 ( LOC_ Os05g48840 ), OsDMP3 ( LOC_Os01g29240 ), OsDMP4 ( LOC_Os02g27800 ) and OsDMP5 ( LOC_ Os12g22270 ). b. Identify positive mutant lines: Mutant lines are self-pollinated to T2 generation, RNA extraction and qRT-RCR analysis are performed, the gene sequence detected by qRT-RCR is subjected to Sanger sequencing, and is compared with the gene sequence of wild type Huahang 48, and if the gene expression is down-regulated and the gene sequence is mutated as expected, it can be determined as a positive mutant line; (3) Identifying the related characteristics of positive mutant lines: Positive mutant lines are self-pollinated, and putative haploids are identified by comparing plant height and leaf characteristics with diploid siblings and wild type (WT) Huahang 48, flow cytometry analysis is used to confirm the ploidy of putative haploids, HIR of selfing is determined, HIR (%)=(number of haploids / total number of plants) x 100%, seed setting rate (SSR) of selfing is calculated, SSR (%)=(number of filled seeds / (number of filled seeds+number of empty seeds)) x 100%, Duncan's new multiple range method is used to analyze the significant difference of mean value of seed setting rate between multiple groups of samples; (4) Identifying the haploid induction ability of positive mutant lines: Positive mutant lines of T2 generation are used as male parents, photoperiod-sensitive sterile line Xian rice line Hang 93S is used as female parent for hybridization, Huahang 48 is used as control male parent, and HIR and SSR are calculated after seed harvest.

2. The method of claim 1, wherein, The prediction method of the structure of the rice haploid induction effect gene is: using Pfam and UniProt websites to predict the protein structure domain of the haploid induction effect related gene, focusing on the motif structure, affinity base active site and proton acceptor active site key structure contained in the gene.

3. The method of claim 2, wherein, The prediction method of the orthologous genes of haploid induction effect genes in rice is: using the full-length amino acid sequence of the identified haploid induction effect genes in other plants for BLASTP, searching the sequence database of rice, selecting genes with amino acid sequence identity >40% and constructing a phylogenetic tree, and analyzing the homologous relationship between the selected genes and the identified haploid induction effect genes in other plants.

4. The method of claim 3, wherein, The detection method of the haploid induction effect related gene expression pattern is: extracting total RNA from the indica rice Huahang 48 plants for mRNA reverse transcription, and performing qRT-PCR on the knocked-out genes, OsUBQ As a reference gene, each sample has 3 biological replicates, each replicate includes 3 technical replicates, and 2 －△△Ct The data is processed by the method to detect the expression pattern of the haploid induction effect related gene.

5. The method of claim 4, wherein, The subcellular localization method of the haploid induction effect related gene is as follows: the protein coding sequence without stop codon is cloned into the SpeI-BamHI site of the PAN580-GFP vector, the recombinant plasmid is co-transformed with mCherry fusion into the protoplast of rice, a control experiment is carried out using a vector without the target gene, the endoplasmic reticulum located mCherry is used as a plasma membrane marker, the nucleus located mCherry is used as a nucleus marker, the vesicle located mCherry is used as a vesicle marker, and FM4-64 is a cell membrane dye; after being cultured at 28 DEG C for 12-16 hours, the green fluorescent signal and the red fluorescent signal are captured.

6. The method of claim 5, wherein, The genes used in creating the double mutant in the method are OsMATL and OsDMP1 or OsMATL and OsDMP3 .

7. The method according to any one of claims 1-6 for use in rice breeding.

8. The use of a haploid induction line of rice selected by the method according to any one of claims 1-6 in rice breeding.

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