Method for creating plant haploid inducer line, allelic mutant of mtl gene and application thereof

Abstract

The application creates new alleles of MTL by a targeted mutagenesis strategy, and is not directly to make MTL lose function, but regulates the expression of MTL in different degrees, or makes individual sequence change, and further improves the fertility and haploid induction rate.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and crop breeding, specifically involving the creation method of plant haploid induction lines and the allelic mutants of the MTL gene and their applications, which can improve the fruit setting rate and induction rate of haploid induction lines. Background Technology

[0002] Efficient breeding of rice (Oryza sativa L.) varieties is a key step in improving rice yield, quality, and resistance. Traditional breeding requires 6-8 generations of continuous self-pollination and selection to obtain relatively stable materials, a lengthy process that is difficult to meet the needs of rice breeding. Double haploid (DH) breeding technology can obtain pure lines in just 2 generations, greatly shortening the breeding process and representing a technological revolution in pure line breeding. The generation of haploids is the first step in double haploid breeding. The frequency of naturally occurring haploids is very low, only 0.1%, which cannot meet the large-scale demand of breeding (Chase SS. Production of homozygous diploids of maize from monoploids. Agron J, 1952, 44: 263–267.).

[0003] In rice, in vitro culture of anthers to induce callus redifferentiation into haploid plants is the main method for producing rice haploids. However, rice anther culture is strictly genotype-dependent and complex to operate. For example, the anther culture ability of indica rice is significantly weaker than that of japonica rice (Hu Jianlin, Zhou Li, Zheng Xingfei, Dong Hualin, Fei Zhenjiang, Zha Zhongping, You Aiqing, Xu Deze. Research status and prospects of in vitro culture of rice anthers. Agricultural Science and Technology Communications, 2019, (12): 57–61). Therefore, the production of rice haploids has always been a bottleneck restricting the breeding of double haploid rice. In recent years, DH technology based on biological induction has been widely used by seed companies and breeding units at home and abroad in maize, becoming one of the three major modern maize breeding technologies, along with molecular marker-assisted breeding and transgenic technology. Biologically induced parthenogenetic haploids are produced by using a haploid inducing line as the male parent and crossing it with the target selection line as the basic material. A certain proportion of maternal bloodline haploids can be obtained in the grains of the hybrid generation. Haploid production using inducing lines has advantages such as not being dependent on genotype, simple induction process, and low cost. In maize, the trait of induced haploid production is controlled by major QTLs, among which qhir1 (MTL gene) can explain 66% of the genetic variation (Chen Shaojiang. Thoughts on Crop Breeding Engineering and Engineered Breeding. Crop Journal, 2013, (6): 1–4.).

[0004] In maize haploid induction lines, a 4 bp (CGAG) insertion in the MTL gene between exon 4 (1572-1573) causes a 20-amino acid frameshift mutation, leading to premature translation termination and haploid induction. MTL is highly conserved in crops, and haploid induction based on MTL gene knockout has been successfully achieved in maize, rice, and wheat using gene editing technology. Syngenta edited exons 1 and 4 of the MTL homolog MTL (Os03g0393900) in indica rice IR58025B, and the mutants induced 2%–6% of haploids. Wang Chun et al. edited the fourth exon of the MTL gene in the indica-japonica hybrid rice Chunyou 84, achieving a haploid induction rate of 4.44% (Wang C, Liu Q, Shen Y, Hua YF, Wang JJ, Lin J R, Wu MG, Sun TT, Cheng ZK, Mercier R, Wang K J. Clonal seeds from hybrid rice by simultaneous genome engineering of meiosis and fertilization genes. Nat Biotechnol, 2019, 37: 283–286). Whether mutations at different sites in the MTL gene in rice affect the haploid induction rate remains to be explored. Furthermore, the influence of different genetic backgrounds of rice materials on the haploid induction rate is also unknown. Therefore, obtaining a high-induction-rate haploid induction line is of great significance for double haploid rice breeding. Wen Qin et al. (Creation and Analysis of MTL Mutants for Haploid Induction in Rice, Acta Agronomica Sinica, 2021, 47(5):827-836) used Nipponbare japonica rice and Huahang 48 indica rice as research materials. They used CRISPR / Cas9 technology to edit multiple target sites in the promoter and coding regions of the MTL gene, obtaining different types of MTL mutants in the context of indica and japonica rice. They further analyzed the mutation effects at different sites, providing basic materials for in-depth research on the mechanism of haploid induction in rice parthenogenesis. In this paper, CRISPR / Cas9 was used to knock out the promoter and coding regions of MTL. Mutants with large deletions in the promoter region had no haploid induction ability, and mutants with multiple knockouts in the coding region all lost MTL function.

[0005] Heterosis is a common phenomenon in the biological world and is widely used in crop breeding and production practices. One of the most important aspects of applying heterosis in agricultural production is the efficient preparation of hybrid seeds. In dioecious crops such as corn, male flowers of the maternal inbred line can be removed manually (or mechanically), and pollination with pollen from another inbred line (paternal parent) can be used to obtain hybrid seeds. This operation is relatively simple and easy to implement, hence the early and mature utilization of heterosis in corn, leading to its widespread application. However, some parents have inconsistent flowering times, making large-scale hybrid seed production impossible in the field. Hermaphroditic crops (such as rice and wheat) cannot achieve large-scale hybrid seed production by removing pollen from the maternal parent. Taking rice as an example: Currently, the approach to solving this problem in rice is to use plants with pollen sterility as the maternal parent and another variety as the paternal parent to provide pollen for hybridization—a heterosis utilization system with male sterility as its core technology. The utilization of hybrid vigor in rice can be divided into two technical approaches: the "three-line method" hybridization technology, which takes nucleocytoplasmic interaction and pollen non-existence as its core technology, and the "two-line method" hybridization technology, which takes photoperiod and temperature-sensitive nuclear male sterility, which is regulated by natural photoperiod and temperature, as its core technology.

[0006] CN109943585B discloses a method using rice as an example, where gene mutation is used to transform the meiotic division of the reproductive cells of a hybrid into a mitotic-like process, thus obtaining gametes with the same genotype and chromosome ploidy as the hybrid; then, the MTL gene is knocked out to induce parthenogenesis, ultimately obtaining cloned seeds. However, inducing haploids by knocking out the MTL gene has two problems: firstly, it affects the seed setting rate, which is only about 10%; secondly, the haploid induction rate is not high, only about 5%. Therefore, further research is needed to explore methods to improve the seed setting rate and induction rate of haploid induction lines. Summary of the Invention

[0007] Previous strategies involved using CRISPR / Cas9 technology to knock out the MTL gene, resulting in the creation of rice haploid inducible lines with lost MTL function. However, these lines exhibited low seed setting and haploid induction rates. Our research has revealed that creating new MTL alleles using a single-base editor does not directly cause MTL function loss. Instead, it regulates MTL expression to varying degrees or alters its spatial structure, thereby improving seed setting and haploid induction rates.

[0008] The inventors analyzed the functional domains of the rice MTL gene and found that it contains only one phospholipase domain (aa32-aa381). Therefore, editing the functional domains of MTL aims to improve the seed setting rate and haploid induction rate of haploid induction lines. A series of sgRNAs targeting the entire phospholipase domain were designed to obtain a series of gene-mutated MTL alleles, enabling directed evolution of the MTL gene. Mutations that improve seed setting rate and haploid induction rate (compared to loss-of-function mutants of MTL) were then screened in the offspring.

[0009] This invention provides a method for creating a plant haploid induction line, characterized by comprising the following steps:

[0010] 1) Target and mutate the protein domain region of MTL, preferably the phospholipase domain region of aa32-aa381, so that the MTL gene in plants undergoes amino acid substitution mutations.

[0011] 2) Obtain plants with the mutant MTL allele;

[0012] 3) Statistically determine the seed setting rate of the obtained MTL allelic plants and detect their haploid induction rate.

[0013] In a specific implementation, the mutation of the MTL gene is achieved using a single base editor (using the CBE single base editor or the adenine base editor ABE), guided editing, HDR-mediated homologous substitution mutation, or by screening after physicochemical mutagenesis.

[0014] The mutation of the MTL gene is achieved by constructing a single-base editor vector containing sgRNA designed to target the sequence on the MTL gene and transforming it into recipient plants.

[0015] Preferably, the transgenic recipient plant is obtained using the gene gun method or the Agrobacterium infection method; more preferably, the single-base editor vector is an Agrobacterium binary transformation vector, which then infects the recipient plant with Agrobacterium. Further, the transgenic plant is obtained by regenerating the callus tissue of the infected recipient plant through Agrobacterium infection.

[0016] Step 2) Obtaining plants with the mutated MTL allele specifically involves planting the plants with the mutated MTL allele (i.e., T0 generation plants), harvesting seeds after maturity, and germinating seedlings to obtain T1 generation plants. Then, screening for lines containing amino acid mutation types. Preferably, the process further includes the following steps: selecting mutants with increased haploid induction rate and obtaining mutant lines through screening for subsequent use.

[0017] In specific embodiments, the plants are monocotyledonous and dicotyledonous plants. More specifically, the plants are selected from rice, corn, sorghum, millet, barley, wheat, rye, oats, buckwheat, Job's tears, sugarcane, asparagus, bamboo shoots, leeks, yams, soybeans, potatoes, peas, mung beans, adzuki beans, broad beans, cowpeas, kidney beans, lentils, mandarins, chickpeas, cassava, sweet potatoes, rapeseed, cotton, beets, eggplant, peanuts, tea, mint, coffee, sesame, sunflower, castor beans, perilla seeds, safflower, tomatoes, peppers, cucumbers, bok choy, lettuce, spinach, garlic, cabbage, mustard greens, water chestnuts, scallions, winter melon, zucchini, loofah, Chinese cabbage, radishes, onions, watermelons, grapes, carrots, cauliflower, pumpkins, tobacco, forage grass, elephant grass, foxtail grass, Sudan grass, orchids, lilies, tulips, and alfalfa. Although rice is provided in the examples, the MTL gene is conserved in other crops, so it is expected to be applicable to the above-mentioned plants as well.

[0018] Further steps include selecting mutants with increased haploid induction rate and obtaining homozygous lines through screening for subsequent use.

[0019] The present invention also provides an allelic mutant of the MTL gene, characterized in that it has one of the following mutations relative to the wild-type MTL gene shown in SEQ ID No. 1:

[0020] R109C

[0021] N108K

[0022] R109W

[0023] N108K,R109W

[0024] R148K

[0025] P166F

[0026] L54F

[0027] A92V

[0028] A93V

[0029] G231N

[0030] V232I

[0031] A92V, A93V

[0032] V232L

[0033] V232I

[0034] D65N

[0035] G66S

[0036] D65N, G66S

[0037] R109C

[0038] N108K

[0039] R109W

[0040] N108K,R109W

[0041] A129V

[0042] R359Q

[0043] A360T

[0044] R359Q, A360T

[0045] R99H.

[0046] The amino acid sequence of the wild-type MTL protein in rice, SEQ ID No. 7, is as follows:

[0047] MAASYSCRRTCEACSTRAMAGCVVGEPASAPGQRVTLLAIDGGGIRGLIPGTILAFLEARLQELDGPDARLADYFDCIAGTSTGGLITAMLAAPGDHGRPLFAASDINRFYLDNGPLIFPQKRCGMAAAMAALTRPRYNGKYLQGKIRKM LGETRVRDTLTNVVIPTFDVRLLQPTIFSTYDAKSMPLKNALLSDICISTSAAPTYLPAHCFQTTDDATGKVREFDLIDGGVAANNPTMVAMTQITKKIMVKDKEELYPVKPSDCGKFLVLSVGTGSTSDQGMYTARQCSRWGIVRWLR.

[0048] This invention further provides the application of the method for creating plant haploid induction lines in plant haploid induction or plant breeding. It also provides the application of allelic mutants of the MTL gene in plant breeding.

[0049] Preferably, the application steps are as follows:

[0050] 1) Using the high-induction-rate haploid induction line obtained by the method for creating plant haploid induction lines described above, or the allelic mutant of the MTL gene described above, as the male parent, and superior hybrids as the female parent, hybridization is performed to obtain haploids, and further haploid doubling is carried out to rapidly breed homozygous inbred lines, which are then used for the combination of superior hybrids; furthermore,

[0051] 2) The haploid induction line, combined with MiMe technology, is used to obtain cloned seeds, thereby achieving heterosis fixation; or

[0052] 3) The haploid induction line is combined with Hi-Edit technology. More specifically, a CRISPR / Cas9 vector for improving agronomic traits is introduced into the haploid induction line through Agrobacterium-mediated transformation to create a haploid induction line carrying the CRISPR / Cas9 vector. Then, the haploid induction line carrying the CRISPR / Cas9 vector is used as the male parent and crossed with any variety to screen for haploids. Then, haploids with edited target sites are screened based on sequencing analysis of the CRISPR / Cas9 target sites. The haploids are doubled into homozygous gene-edited diploid plants through artificial or natural chromosome doubling, thereby realizing the creation of trait-improved commercial rice varieties with double haploids within two generations.

[0053] The plants mentioned are monocotyledonous or dicotyledonous plants, more specifically rice, corn, sorghum, millet, barley, wheat, rye, oats, buckwheat, Job's tears, sugarcane, asparagus, bamboo shoots, leeks, yams, soybeans, potatoes, peas, mung beans, adzuki beans, broad beans, cowpeas, kidney beans, lentils, mandarins, chickpeas, cassava, sweet potatoes, rapeseed, cotton, beets, eggplant, peanuts, tea, mint, coffee, sesame, sunflower, castor beans, perilla seeds, safflower, tomatoes, peppers, cucumbers, bok choy, lettuce, spinach, garlic, cabbage, mustard greens, water chestnuts, scallions, winter melon, zucchini, loofah, Chinese cabbage, radishes, onions, watermelons, grapes, carrots, cauliflower, pumpkins, tobacco, forage grass, elephant grass, foxtail grass, Sudan grass, orchids, lilies, tulips, and alfalfa.

[0054] This invention verifies the reliability of the method for creating haploid inducible lines in plants. Through experiments, the inventors created a series of new alleles of the MTL gene using single-base editing technology. Rice plants carrying these alleles showed varying degrees of change in seed setting rate and haploid induction rate. Compared to rice plants with lost MTL function, some rice plants carrying the new alleles exhibited significantly improved seed setting rate and haploid induction rate. For example, compared to MTL loss-of-function mutants, the currently detected haploid inducible lines carrying MTL alleles showed a certain increase in either seed setting rate or induction rate, with a maximum increase of approximately 10.49 times in seed setting rate and approximately 4.67 times in haploid induction rate. Furthermore, these new MTL alleles are all distributed within the MTL; therefore, amino acid mutations throughout the phospholipase domain all increase the seed setting rate or induction rate of the haploid inducible lines. Attached Figure Description

[0055] Figure 1 MTL structure domain.

[0056] Figure 2MTL 1I MTL N108K,R109W and MTL R109C Plant type diagram of the mutant plant.

[0057] Figure 3 MTL R99H Plant type diagram of the mutant plant. Detailed Implementation

[0058] The present invention will be further illustrated below with specific embodiments to provide a better understanding of the invention, but these embodiments do not constitute a limitation thereof. Specific operations not specifically specified herein are all conventional methods in the art.

[0059] Example 1

[0060] 1. Carrier Construction

[0061] The construction process is described in detail using the construction of amino acid mutations at N108 and R109 of the MTL protein as an example.

[0062] 1)A3A-PBE-g1 MTL Carrier construction

[0063] According to the A3A-PBE system (Zong et al., Nature Biotechnology. 2018) requirements for target sequences: the protospacer adjacent motif (PAM) of the prespacer sequence is NGG, and the sequence length is 22 bp. The following specific target sequences were selected on rice chromosome sequences, with the underlined PAMs:

[0064]

[0065] Based on the sgRNA primer design principles, restriction enzyme ligation bases were added to both ends of the F-direction and R-direction primers, and the designed primers were named g1-F / R. The sequences are as follows:

[0066] g1-F:ggcgACCGCTTCTACCTCGACAA

[0067] g1-R:aaacTTGTCGAGGTAGAAGCGGT

[0068] The designed g1-F / R was annealed at 100℃ for 5 minutes and then allowed to cool naturally. The annealing product was ligated with the BsaI-digested vector A3A-PBE to obtain the ligation product. This ligation product was then transformed into *E. coli* to obtain transformants. The plasmid of the transformants was extracted and sequenced to confirm its correctness, thus obtaining the vector, which was named A3A-PBE-g1. MTL .

[0069] 2) pC1300-UBI-Cas9-g0 MTL Carrier construction

[0070] MTL gene knockout plants were constructed as controls. Following the CRISPR-Cas9 system's target sequence requirements (PAM is NGG, sequence length is 22 bp; specific methods can be found in Chinese patent ZL201510485573.2), the following specific target sequences were selected on the rice chromosome as target sites for MTL knockout:

[0071]

[0072] Based on the principles of sgRNA primer design, restriction enzyme ligation bases were added to both ends of the F-direction and R-direction primers. The designed primers were named g0-F / R. The sequences are as follows:

[0073] g0-F:ggcaGGTCAACGTCGAGACCGGC

[0074] g0-R:aaacGCCGGTCTCGACGTTGACC

[0075] The designed g0-F / R primers were annealed at 100℃ for 5 minutes and then allowed to cool naturally. The annealed product was ligated with the AarI-digested vector pC1300-UBI-Cas9-gRNA to obtain the ligation product. This ligation product was then transformed into *E. coli* to obtain transformants. The plasmid of the transformants was extracted and sequenced to confirm its correctness, yielding the vector, which was named pC1300-UBI-Cas9-g0. MTL .

[0076] 2. Plant transformation: Obtaining transgenic plants

[0077] The A3A-PBE-g1 obtained above MTL and the control group pC1300-UBI-Cas9-g0 MTL Binary vectors were transferred into Agrobacterium tumefaciens EHA105, and then into the callus tissue of the recipient plant, Salt Rice 8, via recombinant bacteria. After culturing at 25°C in the dark for 3 days, resistant transgenic plants were screened on selective medium containing 50 mg / L hygromycin. The screened resistant callus tissue was cultured on predifferentiation medium containing 50 mg / L hygromycin for approximately 10 days. The predifferentiated callus tissue was then transferred to differentiation medium and cultured under light conditions. Resistant transgenic plants were obtained after about one month. These transgenic plants were planted in the field (i.e., T0 generation plants), and seeds were harvested after maturity. Seedlings were then generated to obtain T1 generation rice plants.

[0078] 3. Genotyping and phenotypic identification

[0079] Detection of A3A-PBE-g1 using Hi-TOM gene mutation detection technology MTL and pC1300-UBI-Cas9-g0 MTL Mutation types of resistant transgenic plants, screening for MTL lines with homozygous amino acid mutations. R109C and MTL N108K,R109W and the MTL gene mutant MTL 1I (i.e., converting pC1300-UBI-Cas9-g0) MTL The mutants selected from the gene-edited lines, including MTL 1I This indicates that a T was inserted at position 1425 bp in the MTL genome. Planting was performed and phenotypic observations were conducted; the self-pollination seed set rate was calculated. Figure 2 Display MTL 1I MTL N108K,R109W and MTL R109C Plant architecture diagram of the mutant plant.

[0080] MTL R109C Pollen was used to hybridize the three-line male-sterile rice line Guang A. Haploids were then screened from the resulting hybrid offspring using flow cytometry, and the haploid induction rate was calculated. MTL was then used... N108K,R109W Pollen was used to hybridize the three-line male-sterile rice line Zhongguang A. Haploids were then screened from the resulting hybrid progeny by flow cytometry, and the haploid induction rate was calculated. As a control, MTL was used... 1I Pollen was used to hybridize the three-line male-sterile rice line Guang A. Haploids were then screened by flow cytometry in the resulting hybrid offspring, and the haploid induction rate was calculated.

[0081] The method and procedure for determining plant ploidy by flow cytometry are as follows: Lysis buffer LB01: Tris 363.4 mg, Na2EDTA 148.9 mg, Spermine tetrahydrochloride 34.8 mg, KCl 1.193 g, NaCl 233.8 mg, Triton X-100 200 μL, brought to a final volume of 200 mL, pH adjusted to 7.5 with 1 M HCl, and 220 μL of β-mercaptoethanol added in a fume hood. The mixture was sterilized and dispensed using a 0.22 μm filter in a clean bench and stored at -20 °C.

[0082] Propidium iodide (PI) stock solution (1 mg / ml): Weigh 50 mg of powder and dissolve it in 50 mL of ddH2O; sterilize and dispense the solution by filtration through a 0.22 μm filter in a clean bench and store at -20 °C.

[0083] RNase stock solution (1 mg / ml): Weigh 25 mg RNase (IIA Sigma) and dissolve it in 25 mL ddH2O; sterilize and dispense by filtration using a 0.22 μm filter in a clean bench; heat at 90 °C for 15 min to inactivate DNase; store at -20 °C.

[0084] The specific procedure is as follows: Cut fresh rice leaves (4-5 cm long) that have grown for 10 days and place them in a glass dish. Add 1 mL of plant lysis buffer LB01 and quickly mince the tissue vertically downwards with a blade (this operation should always be performed on ice). Aspirate the lysis buffer from the culture dish and filter it through a 50 μm nylon mesh into a centrifuge tube. Label the tube cap with the sample. Centrifuge at 1,200 rpm for 5 min at 4°C in a benchtop refrigerated centrifuge. Gently remove the centrifuge tube, slowly aspirate the supernatant, and add 450 μL of LB01, 25 μL of pre-chilled PI, and 25 μL of RNase A (a mixture can be prepared in advance). Stain at 4°C in the dark for 10 min. Analyze using BD Accuri C6 chromatography.

[0085] Seed setting rate statistics: The seed setting rate of wild-type salt rice '8' was 83.73±3.31%, and the haploid induction rate was 0%; the control MTL... 1I The seed setting rate was 6.93±2.03%, and the haploid induction rate was 4.35%; MTL R109C The seed setting rate was 54.88±15.33%, and the haploid induction rate was 19.44%; MTL N108K,R109W The seed setting rate was 63.26 ± 0.96%, and the haploid induction rate was 18.07%. Compared to the control MTL... 1I In terms of MTL R109C and MTL N108K，R109W Significant improvements were achieved in both seed setting rate and haploid induction rate. Therefore, mutating the MTL gene can increase both seed setting rate and haploid induction rate.

[0086] Following the same strategy and methods described above, single-base mutations at various other sites were obtained (see Table 1). Similarly, the constructed vector was transformed into rice callus tissue using Agrobacterium-mediated transformation. Subsequently, the obtained transgenic positive lines were subjected to mutation detection using Hi-TOM technology. Multiple homozygous gene-edited lines with mutations were identified and screened, and their seed setting rates were calculated. Simultaneously, a three-line sterile line was selected as the female parent, and the homozygous gene-edited line with mutations was selected as the male parent for hybridization. The haploid induction rate was calculated (results are shown in Table 1).

[0087] Table 1. Statistics on self-pollination seed setting rate and haploid induction rate of homozygous mutant lines.

[0088]

[0089]

[0090] As shown in Table 1, the self-crossing seed setting rate or hybridization haploid induction rate of these haploid induction lines carrying the new MTL alleles were higher than those of the MTL loss-of-function mutant. Among them, the MTL alleles were the most prominent. N108K，R109W For example, its self-pollination seed setting rate increased by approximately 9.13 times, based on MTL. R109C For example, its haploid induction rate increased by approximately 4.47 times. Note that in cases showing an induction rate of 0, the mutation may have only affected the spatial structure of the MTL, but still did not enable it to induce haploids.

[0091] Example 2

[0092] 1. Obtaining mutagenic materials

[0093] The conventional rice variety Yandao 8 was treated with EMS mutagenesis, and the M0 generation Yandao 8 after EMS mutagenesis was sown.

[0094] 2. Genotyping and phenotypic identification

[0095] DNA was extracted from single M0 leaf plants, and MTL mutant plants were screened using Hi-TOM high-throughput sequencing technology. MTL-carrying plants were successfully identified. R99H A mutant plant was obtained. This plant was planted, and its seeds were harvested after maturity to obtain offspring M1. M1 plants were then planted to obtain MTL. R99H Phenotypic observation was conducted on homozygous mutant plants at the locus, and their self-pollination seed setting rate was statistically analyzed. Figure 3 Display MTL R99H Plant architecture diagram of the mutant plant.

[0096] MTL R99H Pollen was used to hybridize the three-line male-sterile rice line Zhongguang A. The hybrid offspring were further screened for haploids and double haploids by genetic molecular markers (those that only had the Zhongguang A marker in the maternal parent were identified as haploids or double haploids). Flow cytometry and whole genome sequencing were then used to determine the haploid induction rate.

[0097] The method and procedure for identifying plant ploidy by flow cytometry are the same as in Example 1.

[0098] Seed setting rate statistics: The seed setting rate of wild-type salt rice '8' was 85.21±3.28%, and the haploid induction rate was 0; MTL R99H The seed setting rate was 40.93 ± 8.33%, and the haploid induction rate was 11.2%; compared with the control MTL in Example 1. 1I In terms of MTL R99HThe experiment significantly improved both the seed setting rate and the haploid induction rate. This further demonstrates that mutating the MTL gene can increase both seed setting rate and haploid induction rate, and that these goals can be achieved using gene editing or other mutagenesis methods.

[0099] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention. sequence list <110> China Rice Research Institute <120> Methods for creating plant haploid inducible lines and allelic mutants of the MTL gene and their applications <160> 7 <170> Patent-In 3.3 <210> 1 <211> twenty two <212> DNA <213> Artificial sequence <400> 1 accgcttctacctcgacaacgg 22 <210> 2 <211> twenty two <212> DNA <213> Artificial sequence <400> 2 ggcgaccgcttctacctcgacaa 23 <210> 3 <211> twenty three <212> DNA <213> Artificial sequence <400> 3 aaacttgtcgaggtagaagcggt 23 <210> 4 <211> twenty three <212> DNA <213> Artificial sequence <400> 4 ggcaggtcaacgtcgagaccggc 23 <210> 5 <211> twenty three <212> DNA <213> Artificial sequence <400> 5 aaacgccggtctcgacgttgacc 23 <210> 6 <211> 22 <212> DNA <213> Artificial sequence <400> 6 ggtcaacgtcgagaccggcagg 22 <210> 7 <211> 22 <212> PRT <213> Artificial sequence <400> 7 MAASYSCRRTCEACSTRAMAGCVVGEPASAPGQRVTLLAIDGGGIRGLIPGTILAFLEARLQELDGPDARLADYFDCIAGTSTGGLITAMLAAPGDHGRPLFAASDINRFYLDNGPLIFPQKRCGMAAAMAALTRPRYNGKYLQGKIRKMLGETRVRDTLTNVVIPTFDVRLLQPTIFSTYDAKSMPLKNALLSDICISTSAAPTYLPAHCFQTTDDATGKVREFDLIDGGVAANNPTMVAMTQITKKIMVKDKEELYPVKPSDCGKFLVLSVGTGSTSDQGMYTARQCSRWGIVRWLR 299

Claims

1. A kind MTL Allelic mutants of a gene, characterized by, The MTL The protein encoded by the allelic mutant of the gene has only the following mutations with respect to the wild-type protein shown in SEQ ID No. 7 MTL The protein encoded by the allelic mutant of the gene has only the following mutations with respect to the wild-type protein shown in SEQ ID No. 7 (1) L54F; (2) R109C; (3) R109W; or (4) N108K and R109W.

2. The method of claim 1, wherein the plant is a rice plant. MTL Use of allelic mutants of a gene in plant haploid induction or plant breeding, the plant being a rice plant.

3. Use according to claim 2, wherein the compound is ###0002### The application steps are as follows: 1) rice plants comprising the allele of the gene as claimed in claim 1 MTL Rice plants comprising the allele of the gene as claimed in claim 1 are crossed with elite hybrids as female parents, haploids are obtained and further doubled to obtain homozygous inbred lines which are further used for the development of elite hybrids.

4. The use according to claim 3, wherein the compound is ###0002### The haploid doubling is to artificially or naturally double the haploid chromosome to become a homozygous genetically edited diploid plant.

Images